Treatment of vascular dysfunction and alzheimer&#39;s disease

ABSTRACT

Dysregulation of vascular function is observed in brain endothelium derived from patients affected by Alzheimer&#39;s disease. This may be manifested at the cell or organ level: e.g., abnormal capillary morphogenesis, defective angiogenesis, inappropriate cellular senescence, mitotic catastrophe, or combinations thereof. This observation can be used for diagnosis or treatment of neurodegenerative disorders and other cognitive impairments.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of provisional Appln. No.60/387,426, filed Jun. 11, 2003; Appln. No. 60/387,427, filed Jun. 11,2003; and Appln. No. 60/387,913, filed June 13, 2003. The contents ofthese provisional patent applications and Appln. No. PCT/US02/01069 areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to neurodegenerative disorders and cognitiveimpairments (e.g., Alzheimer's disease) and the dysregulation ofvascular function which is observed in brain endothelial cells (BEC)derived from patients.

BACKGROUND OF THE INVENTION

Brain degenerative diseases are associated with dysfunction of learning,memory, and/or cognition include cerebral senility, multi-infarctdementia, senile dementia of the Alzheimer type, age-associated memoryimpairment, and certain disorders associated with Parkinson's disease.Alzheimer's disease is the most common of the age-relatedneurodegenerative diseases: between about 10% and 20% of individualsover age 70 are affected, and about 50% of those over age 85 areaffected. It is estimated that about 50% of nursing home residents inthe U.S. are affected, and that the annual costs associated with thecare of patients with Alzheimer's disease in this country are in excessof $65 billion. As the population ages, the prevalence of Alzheimer'sdisease will increase dramatically from four million presently in theU.S. to more than 10 million by 2015. Study of the molecular basis ofAlzheimer's disease complements behavioral studies. It can lead to abetter understanding of pathogenesis and mechanisms of disease, as wellas new modes of treatment.

Current dogma teaches that many different initiating events willultimately cause synapses to fail to function properly and this leadsinexorably to neuronal death. Several neuropathological findings areassociated with Alzheimer's disease and the following are consideredindicia of the.Alzheimer's phenotype: intraneuronal deposits ofneurofibrillary tangles (NFT), parenchymal amyloid deposits—neuriticplaques, cerebral amyloid angiopathy (CAA), and synaptic loss. Popularcurrent theories for the cause of Alzheimer's disease are the amyloid,tau, and inflammatory theories. Mutations in three genes encodingamyloid-β precursor protein (APP), presenilin-1, and presenilin-2 causerare, early-onset, autosomal dominant forms of Alzheimer's disease.These mutations all affect APP metabolism such that more amyloid-β (AP)peptide is produced. In contrast, most cases of Alzheimer's disease haveages of onset above 65 years and exhibit no clear pattern of inheritance(i.e., late onset Alzheimer's disease or LOAD). The E4 allele of theapolipoprotein E (apoE) gene is the only known risk factor for LOAD. But50% of late-onset cases carry no apoE4 alleles, which indicates thatthere must be additional risk factors. Recent studies have identifiedthe locus for LOAD on chromosome 10 and linked it with increased levelsof circulating Aβ₁₋₄₂. See refs. 1-10.

Deposition of Aβ in the CNS occurs during normal aging and isaccelerated by Alzheimer's disease. Aβ is implicated in theneuropathology of Alzheimer's disease and related disorders. Recentstudies suggest that the BBB plays a major role in determining theconcentration of Aβ in the CNS. The BEC at the BBB have a dual role: (a)to control entry of plasma-derived Aβ and its binding/transport proteinsinto the CNS, and (b) to regulate levels of brain-derived Aβ viaclearance mechanisms. See refs. 11-22.

Previous genetic and biochemical approaches neither taught nor suggestedthat Alzheimer's disease is associated with or may be caused bydisease-specific mRNA and protein profiles in BEC related to altered BECbiology distinct from normal aging, that could be manifested at theorgan level as abnormal responses to angiogenic stimulation, aberrantformation of brain capillaries, formation of incompetent brain capillarynetworks, accelerated and/or premature removal of BEC from the vascularsystem during capillary morphogenesis through cell death programs (e.g.,apoptosis, aniokis, mitotic catastrophe), and development of a senescentphenotype resulting in loss of regulatory functions of the BBB and CBF(cf. St. George-Hyslop, Sci. Am. pp. 76-83, December 2000). Inparticular, it was only recently described that dysfunction of brainendothelium may cause and/or be the result of disease (Int'l PatentAppln. PCT/US02/01069).

These observations can be used to improve our understanding of thepathogenesis of Alzheimer's disease and mechanisms of disease. Novel andinventive methods of diagnosis and treatment are suggested by them.Other advantages of the invention are discussed below or would beapparent from the disclosure herein.

References

1. Wisniewski et al. (1997) Biology of Aβ amyloid in Alzheimer'sdisease. Neurobiol. Dis. 4:311-328.

2. Selkoe (1997) Alzheimer's disease: Genotype, phenotype, andtreatments. Science 275:630631.

3. Selkoe (1998) The cell biology of beta-amyloid precursor protein andpresenilin in Alzheimer's disease. Trends Cell Biol. 8:447453.

4. Younkin (1998) The role of A beta 42 in Alzheimer's disease. J.Physiol. (Paris) 92:289-292.

5. Roses (1998) Alzheimer disease: A model of gene mutations andsusceptibility polymorphisms for complex psychiatric diseases. Amer. J.Med. Gen. 81:49-57.

6. Hardy et al. (1998) Genetic dissection of Alzheimer's disease andrelated dementias: Amyloid and its relationship to tau. Nature Neurosci.1:355-358.

7. Dickson (1997) The pathogenesis of senile plaques. J. Neuropathol.Exp. Neurol. 56:321-339.

8. Blacker et al. (1998) Alpha-2 macroglobulin is genetically associatedwith Alzheimer disease. Nature Gen. 19:357-360.

9. Ertekin-Taner et al. (2000) Linkage of plasma Aβ42 to a quantitativelocus on chromosome 10 in late-onset Alzheimer's disease pedigrees.Science 290:2303-2304.

10. Myers et al. (2000) Susceptibility locus for Alzheimer's disease onchromosome 10. Science 290:2304-2305.

11. Zlokovic (1997) Can blood-brain barrier play a role in thedevelopment of cerebral amyloidosis and Alzheimer's disease pathology.Neurobiol. Dis. 4:23-26.

12. Zlokovic et al. (1993) Blood-brain barrier transport of circulatingAlzheimer's amyloid β. Biochem. Biophys. Res. Commun. 197:1034-1040.

13. Maness etal. (1994) Passage of human amyloid-β protein 1-40 acrossthe murine blood-brain barrier. Life Sci. 55:1643-1650.

14. Poduslo et al. (1997) Permeability and residual plasma volume ofhuman, Dutch variant, and rat amyloid β-protein 1-40 at the blood-brainbarrier. Neurobiol. Dis. 4:27-34.

15. Ghilardi et al. (1996) Intra-arterial infusion of [¹²⁵I]Aβ 1-40labels amyloid deposits in the aged primate brain in vivo. Neuroreport7:2607-2611.

16. Mackic et al. (1998) Cerebrovascular accumulation and increasedblood-brain barrier permeability to circulating Alzheimer's amyloid-βpeptide in aged squirrel monkey with cerebral amyloid angiopathy. J.Neurochem. 70:210-215.

17. Zlokovic et al. (1996) Glycoprotein 330/megalin: Probable role inreceptor-mediated transport of apolipoprotein J alone and in a complexwith Alzheimer's disease amyloid β at the blood-brain andblood-cerebrospinal fluid barriers. Proc. Natl. Acad. Sci. USA93:4229-4236.

18. Martel et al. (1997) Isoform-specific effects of apolipoproteins E2,E3, E4 on cerebral capillary sequestration and blood-brain barriertransport of circulating Alzheimer's amyloid β. J. Neurochem.69:1995-2004.

19. Shibata et al. (2000) Clearance of Alzheimer's amyloid-beta 1-40peptide from brain by low-density lipoprotein receptor-related protein-1at the blood brain barrier. J. Clin. Invest. 106:1489-1499.

20. Mackic et al. (1998) Human blood-brain barrier receptors forAlzheimer's amyloid-β 1-40: Asymmetrical binding, endocytosis andtranscytosis at the apical side of brain microvascular endothelial cellmonolayer. J. Clin. Invest. 102:734-743.

21. Zlokovic (1996) Cerebrovascular transport of Alzheimer's amyloid-βand apolipoproteins J and E: Possible anti-amyloidogenic role of theblood-brain barrier. Life Sci. 59:1483-1497.

22. Ghersi-Egea et al. (1996) Fate of cerebrospinal fluid-borne amyloidβ-peptide: Rapid clearance into blood and appreciable accumulation bycerebral arteries. J. Neurochem. 67:880-883.

SUMMARY OF THE INVENTION

In one embodiment of the invention, reagents are provided in kit formthat can be used for performing the methods such as the following:diagnosis, identification of those at risk for disease or alreadyaffected, or determination of stage of disease or its progression. Inaddition, the reagents may be used in methods related to the treatmentof disease such as the following: evaluation whether or not it isdesirable to intervene in the disease's natural history, alteration ofthe course of disease, early intervention to halt or slow progression,promotion of recovery or maintenance of function, provision of targetsfor beneficial therapy or prophylaxis, comparison of candidate drug,medical, or surgical regimens, or determination of the effectiveness ofa drug, medical, or surgical regimen. The instructions for performingthese methods, reference values and positive/negative controls, andrelational databases containing patient information (e.g., genotype,medical history, symptoms, transcription or translation yields from geneexpression, physiological or pathological findings) are other productsconsidered to be aspects of the invention.

In other embodiments of the invention, the methods for diagnosis andtreatment are provided. For screening of drugs and clinical trials, therespective drug and medical/surgical regimen selected are alsoconsidered to be embodiments of the invention. The amount and length oftreatment administered to a cell, tissue, or individual in need oftherapy or prophylaxis is effective in treating the affected cell,tissue, or individual. One or more properties/functions of affectedendothelium or cells thereof, or the number/severity of symptoms ofaffected individuals, may be improved, reduced, normalized, ameliorated,or otherwise successfully treated. The invention may be used alone or incombination with other known methods. Instructions for performing thesemethods, reference values and positive/negative controls, and relationaldatabases containing patient information are considered further aspectsof the invention. The individual may be any animal or human. Mammals,especially humans and rodent or primate models of disease, may betreated; thus, both human and veterinary treatments are contemplated.

Further aspects of the invention will be apparent to a person skilled inthe art from the following detailed description and claims, andgeneralizations thereto.

DESCRIPTION OF THE TABLES AND DRAWINGS

FIG. 1 shows brain capillary morphogenesis mediated by control and ADBEC in 3-D collagen matrices after stimulation with VEGF/bFGF (40 ng/ml)using an assay system as reported (Davis et al., J. Cell Sci.114:917-930, 2001). FIGS. 1A-1C show formation of brain capillaries fromage-matched control BEC: (A) the formation of intracellular vacuoles at4 hr (arrow, bar 20 μm); (B) vacuoles at 15 hr (arrows, toluidinestaining, bar 25 μm); and (C) capillary tubes at 24 hr (arrows, Hoechststaining, bar 12 μm). FIGS. 1D-1F show aberrant brain capillaryformation mediated by AD BEC: (D) apoptotic bodies (arrow), condensedchromatin, and/or 10 fragmented nuclei (asterisk, Hoechst, bar 12 μm);(E) blebbing of the cytoplasmic membrane (bar 12 μm); and (F) nuclearfragmentation (asterisk; Hoechst, 12 μm) and apoptotic bodies (arrows)at 24 hr.

FIG. 2 summarizes quantitative data on time course of formation ofintracellular vacuoles (stage I) and tubes (stage II) during braincapillary morphogenesis mediated by AD or control BEC after stimulationwith VEGF/bFGF as in FIG. 1. FIG. 2A shows the percentage of cellsforming vacuoles was calculated as a fraction of total number of cells.FIG. 2B shows the number of tubes and FIG. 2C shows the total tubelength determined at 24 hr. Values are mean±s.e. from 12 to 20measurements derived from six cases for each studied group; each casewas tested in triplicate or greater. Significance was evaluated byStudent's t-test.

FIG. 3 shows apoptosis during BEC differentiation into brain capillarytubes in AD model studied in 3-D collagen gel cultures. TUNEL positivecells (bar=15 μm) with nuclear changes (Hoechst) are seen in AD BECafter stimulation with VEGF/bFGF at 4 hr (FIGS. 3A-3B); no such changeswere observed in age-matched BEC (FIGS. 3C-3D). FIG. 3E shows the numberof TUNEL positive cells at 4 hr. Mean±s.e., from 6 cases per groupdetermined for each case in 8 to 10 different fields; *p<0.01, AD vs.controls by Student t-test. Western blot analyses of cell lysates forp53 and activated form of caspase 3 at 4 hr (FIG. 3F), 12 hr (FIG. 3G),and 24 hr (FIG. 3H) during brain capillary morphogenesis. Relativeabundance of p53 (FIG. 3I) and caspase-3 (FIG. 3J) normalized to β-actinin young and age-matched controls and AD. The relative levels ofexpression of p53 and caspase-3 in young controls were arbitrarily setas 1 for each studied time point. Mean±s.e., from 4 to 5 cases; *p<0.01,AD vs. controls by Student t-test. VEGF and bFGF, 40 ng/ml.

FIG. 4 shows that zVAD, a broad caspase-3 inhibitor, restores tubeformation during AD BEC-mediated brain capillary angiogenesis. zVAD-fmk(50 μM) reduced the levels of activated form of caspase-3 in AD BEC at24 hr (FIG. 4A), and restored the number of tubes (FIG. 4B) and totaltube length (FIG. 4C) during AD BEC-mediated morphogenesis. Mean±s.e.,from 4 cases per group; significance by Student t test. VEGF and bFGF,40 ng/ml.

FIG. 5 shows increased expression of p53 and caspase 3 in brainmicrovessels in situ in patients with AD compared to age-matchedcontrols, thus corroborating our findings in BEC model (FIG. 3). Doublestaining for collagen IV (vascular basement membrane marker) and p53 inBrodmann's areas 9 and 10 in AD (FIGS. 5A-5B) or age-matched controls(FIGS. 5C-5D) was performed. Double staining for collagen IV and theactive form of caspase-3 in Brodmann's areas 9 and 10 in AD patients(FIGS. 5E-5F) or age-matched controls (FIGS. 5G-5H) was performed. Thenumber of p53-positive vessels (FIG. 5I) and caspase 3-positive vessels(FIG. 5J) relative to collagen IV in areas 9 and 10 in AD andage-matched controls was calculated. Mean±s.e., n=6 cases per group.

FIG. 6 shows that activated protein C (APC), recently shown to exhibitsignificant anti-apoptoic activity in BEC during hypoxia/ischemia (Chenget al., Nature Med. 9:338-342, 2003), improves tubule formation,prevents apoptosis of AD BEC, and enhances their migration. AD orage-matched control (AMC) BEC (2×10⁴ cells/well) were plated in 2-Dcollagen matrigels (growth factors per BD Biosciences, Bedford, Mass.)and treated with 100 nM APC or vehicle (−APC). Total tube length wasdetermined at 24 hr (FIG. 6A). Mean±s.e., n=30 to 40 assays per groupfrom four AD and three AMC cases. AD or AMC BEC were treated with 100 nMAPC, 100 nM boiled (B) APC, 100 nM serine mutant (M) APC, or vehicle(−APC) for 24 hr. Total tube length was determined at 24 hr (FIG. 6B).Mean±s.e., n=10 assays per group from one AD and one AMC case. %TUNEL-positive BEC at 4 hr during AD-mediated or AMC-mediated capillarymorphogenesis in the presence of 100 nM APC or vehicle (−APC) wascalculated (FIG. 6C). Mean±s.e., n=20 assays per group from two AD andtwo AMC cases. % Caspase-3-positive BEC at 4 hr of brain capillarymorphogenesis in AD or AMC treated with 100 nM APC or vehicle (−APC) wascalculated (FIG. 6D). Mean±s.e., n=20 assays per group from two AD andtwo AMC cases. AD BEC (3×10⁴) cells were assayed for migration in amodified Boyden chamber in the presence of vehicle (−APC), 100 nM APC,anti-APC C3 monoclonal antibody (200 μg/ml), or anti-APC+APC after 6 hrof incubation (FIG. 6E). Mean±s.e., n=6 assays per group from two ADcases. Migration of AD BEC was also assayed in the presence of vehicle(−APC), 100 nM APC, anti-EPCR antibody raised against the APC bindingsite (Dr Fukudome; 200 μg/ml), or anti-EPCR+APC (FIG. 6F). Mean±s.e.,n=6 assays per group from two AD cases.

FIG. 7 shows impaired cell growth of AD BEC compared to age-matchedcontrol brains and the presence of enlarged β-galactosidase positivecells with flattened morphology. FIG. 7A shows growth curves for AD BEC(n=3) and age-matched control (AMC) BEC (n=4). The culture wasmaintained subconfluent for 5 days. The cells in triplicate wells werecounted daily (mean±s.e.m.). Population doubling time (PDT) in AD wascompared to AMC (p<0.01) in FIG. 7B. Accelerated senescence of AD BEC isshown by senescence-associated β-galactosidase (<-gal) assay of culturedBEC for AD at PD 23 (FIG. 7C) and PD 34 (FIG. 7D), and AMC at PD 44(FIG. 7E). Bar=50 μm. Total replicative potential was expressed as CPDL(cumulative population doubling life) in AD BEC (n=8) compared in FIG.7F to AMC BEC (n=5).

FIG. 8 shows the H₂O₂ model of stress-induced premature senescence(SIPS) in young human BEC that result in a cellular phenotype similar tothat seen in replicative BEC senescence of AD. Subconfluent BEC fromyoung humans were treated with 300 μM of H₂O₂ for 2 hr followed bysubculture. Growth arrest in untreated control BEC (FIG. 8A) or SIPS BEC(FIG. 8B) was determined by BrdU incorporation and FACS analysis.Morphological change and SA-β-galactosidase activity typical forsenescent phenotype five days after H₂O₂ treatment was shown inuntreated control BEC (FIG. 8C) or SIPS BEC (FIG. 8D). Bar=50 μm.Phosphorylation of p38 and p53, and induction of p53 and p21 in responseto H₂O₂ was determined by Western blot analyses (FIG. 8E, time inminutes). Activation of p53, p21, and p16 over 5 days afterH₂O₂-treatment was shown by Western blot analyses (FIG. 8F, time indays).

FIG. 9 shows upregulation of p16 during replicative or stress-inducedsenescence in AD BEC cultures. Western blot analysis of p16 in AD BECcultures from passage 5-7 (FIG. 9A, case numbers are indicated abovepassage numbers) and signal intensity for p16 normalized for β-actin(FIG. 9C, n=4) are shown. H₂O₂ stress-induced senescence increases p16over six days (FIG. 9B, case numbers are indicated above time in days)and signal intensity normalized for β-actin (FIG. 9D, n=5) are shown.

FIG. 10 confirms increased number of p16-positive brain microvessels inAD (A-C) compared to age-matched controls (D-F), thus corroboratingfindings in the in vitro BEC model. Brain sections from Brodmann's area9 or 10 were stained with antibodies to Von Willebrand factor (FIGS. 10Aand 10D) and p16 (FIGS. 10D and 10E) as well as merged images (FIGS. 10Cand 10F). The number of p16-positive microvessels was scored for threeAD and three AMC cases (FIG. 10G, bar=50 μm).

FIG. 11 shows that p38 MAPK inhibitor SB202190 prevents BEC-mediatedaberrant angiogenesis caused by either stress-induced prematuresenescence (SIPS) in control cells or in AD BEC. In 3D collagen gels (asdescribed in FIG. 1), BEC that reached greater than 90% replicativesenescence (RS) or were exposed to H₂O₂ (SIPS) formed an insignificantnumber of tubes (FIGS. 11A-11B, bar=50 μm). Total tube length—controlBEC (arbitrarily taken as 100%) were compared to RS and SIPS BEC (FIG.11C). The effect of SB202190 (SB, 10 μM for 1 hr) on SIPS-inducedaberrant angiogenesis. Mean±s.e., n=9 assays per group from threedifferent cases/group. In 2D collagen matrigels (growth factors per BDBiosciences, Bedford, Mass.), SB202190 (SB, 10 μM) increased the numberof tubes in early passage AD BEC (FIGS. 11D-11E, bar=50 μm). SB202190(SB, 10 μM) increases total tube length in both AMC and AD BEC (FIG.11F). Mean±s.e., n=6-9 assays per group from two AMC and three AD cases.

Tables 1-8 summarize characteristics of patients and controls used fordifferent types of studies including gene expression profiling onAffymetrix U95A, U133A, and U133B chips, and for senescence studies.

Tables 9-12 summarize changes in gene expression in BEC AD vs.age-matched controls using Affymetrix U95A, U133A, and U133B chips, andcoincidence analysis of genes in RS-AD and SIPS-YC suggesting that SIPSmodel has significant features of BEC senescence AD-type.

DETAILED DESCRIPTION OF THE INVENTION

The dysregulation of vascular function which is observed in brainendothelial cells (BEC) derived from patients with Alzheimer's disease(AD) is not related to previously observed pathology like that involvedin production of amyloid (i.e., synthesis of amyloid precursor proteinand its processing and metabolism). Such dysregulation may be manifestedat the cell or organ level as abnormal BEC differentiation in responseto angiogenic signaling, activation of a programmed cell death throughapoptosis and/or other forms of death (e.g., anoikis, mitoticcatastrophe) during brain capillary morphogenesis, development ofpremature cellular senescence, or combinations thereof. The alteredbiology of BEC derived from AD patients is associated with, orcontributes to and/or may result from a disease-specific gene expressionprofiles associated with changes in expression of genes with predictedactions in cell differentiation, angiogenesis, signal transduction,cytoskeleton, matrix, lipid metabolism, etc., largely independent ofnormal aging. These related molecular abnormalities in AD BEC ultimatelylead to a failure of successful brain vascular repair in AD (e.g.,aberrant formation of new incompetent brain capillaries) and/or vascularsenescence with altered BEC phenotype and greatly diminished braincapillary functions. These observations can be used to diagnose avascular disorder in AD that clinically may present as abnormalities incerebral blood flow (CBF) and blood-brain barrier (BBB) regulatoryfunctions associated with mild cognitive impairment (MCI) inasymptomatic individuals or dementia in symptomatic individuals, toidentify those at risk for the vascular disease AD type or those alreadyaffected thereby, to determine the stage of the disease or the disease'sprogression, to intervene earlier in or alter the disease's naturalhistory, to provide targets for therapeutic or prophylactic treatments,to screen drugs or compare medical regimens, to determine theeffectiveness of a drug or medical regimen in treating the disease, orany combination thereof. Examples of drug-based therapies for ADvascular disorder involving small molecules and proteins are reported.

These studies are distinguished from previous neural and vasculartheories for explaining the etiology and pathogenesis of Alzheimer'sdisease because they focus on the roles of BEC differentiation, brainvascular repair, and abnormal angiogenesis, on the one hand, andcellular senescence of BEC, on the other, in promoting dysfunction ofthe vascular endothelium and vascular disorder in Alzheimer's disease.Furthermore, these studies link for the first time a subset ofdisease-specific genes (e.g., 0.3% of 12,500 genes using Affymetrix U95Achips; 0.2-0.3% of 45,000 genes using Affymetrix U133A and U133B chips)to abnormalities in AD BEC biology as demonstrated by an in vitro AD BECmodel and corroborated by the analysis of brain vessels in AD tissue insitu. Endothelial cells of brain microvessels, which are derived mainlyfrom capillaries (about 90% to 95%) and a small percentage (about 5 to10%) originating from smaller venuies and arterioles (less than 20 μmdiameter), have been studied. A role for dysregulation of vascularfunction is demonstrated herein which differs from the previous vasculartheories of AD, centered on changes in circulating Aβ transport throughthe BBB: e.g., apoj-, apoE-, RAGE- (Zlokovic et al., Proc. Natl. Acad.Sci. USA 93:4229-4236, 1996; Martel et al., J. Neurochem. 69:1995-2004,1997; Mackic et al., J. Clin. Invest. 102:734-743, 1998; Deane et al.,Nature Med., in press); and/or LRP-1-mediated clearance of Aβ from thebrain (Zlokovic et al., Nature Med. 6:718-719, 2000; Shibata et al., J.Clin. Invest. 106:1489-1499, 2000; Zlokovic et al., In: Aβ Metabolism inAlzheimer's Disease, Ed. T. Saido, Landes Bioscience, pp. 114-122,2003), clearance by peripheral Aβ-plasma binding agents includinganti-Aβ antibodies (DeMattos et al., Science 295:2264-2267, 2002),gelsolin and gangli-oside M2 (Matsuoka et al., J. Neurosci. 23:29-33,2003), sRAGE (Deane et al., Nature Med., in press), sLRP-1 clusters 11and IV (Zlokovic et al. Soc. Neurosci. Abstract No. 1974, in press)and/or permeabilizers of the BBB such as insulin-like growth factor-1(Carro et al., Nature Med. 8:1390-1397, 2002); association of amyloidwith blood vessels and early onset familial form of cerebral amyloidangiopathy (CAA) (Levy et al., Science 248:1124-1126, 1990; VanBroeckhoven et al., Science 248, 1120-1122, 1990; Haass et. al., J.Biol. Chem. 269:17741-17748, 1994; Grabowski et al., Ann. Neurol.49:697-705, 2001; De Jonghe et al., Neurobiol. Dis. 5:281-286, 1998;Hendricks et al., Nature Genet. 1:218-221, 1992; Kamino etal., Am. J.Hum. Genet. 51:998-1014, 1992; Tagliavini et al., Alzheimer's Reports 2,S28, 1999); vasoconstrictory effects of Aβ on blood vessels andproinflammatory and vasoactive effects of Aβ in the cerebrovasculature(Thomas et al., Nature 380:168-171, 1996), Paris et al., Neurobiol.Aging 21, 183-197, 2000; Townsend et al., Ann. NY Acad. Sci. 977:65-76,2002; Volmar et al., Soc. Neurosci. Abstract No. 882.1, 2002); cerebralendothelial dysfunction in mice overexpressing Aβ precursor protein thatcan be rescued by reactive oxygen species scavengers (ladecola et al.,Nature Neurosci. 2:157-161, 1999); Aβ-related functional hyperemia andchanges in CBF in Alzheimer's mouse model (Niwa et al., Proc. Natl.Acad. Sci. USA 97:9735-9740, 2000; Niwa et al., Neurobiol. Dis. 9:6168,2002); brain capillary distortions and microvascular aberrationsdetected in brains of individuals with Alzheimer's disease by light orelectron microscopy (Miyakawa et al., Virchows Arch. 40:121-129, 1982;Yamada et al., Dement. Geriatr. Cogn. Dis. 8:163-168, 1997, Grammas etal., J. Alzheimer's Dis. 4:217-223, 2002); reported risks forAlzheimer's disease compiled from epidemiological studies of elderlypatients with reduced cerebral perfusion and/or risk factors associatedwith both Alzheimer's disease and vascular dementia (Breteler et al.,Neurobiol. Aging 21:153-160, 2000; Hofman etal., Lancet 349:151-154,1997; see for review de la Torre, Stroke 33:1152-1162, 2002; Zlokovic,Adv. Drug Deliv. Rev. 54:1533-1537, 2002; Zlokovic, Adv. Drug Deliv.Rev. 54:1553-1559, 2002; Rotterdam Scan Study, New Engl. J. Med.348:1215-1222, 2003).

Endothelial cells and cultures thereof from brain (e.g., brainmicrovasculature, leptomeningeal vessels) or possibly other organs(e.g., bone marrow, blood containing endothelial precursor cells,extracranial blood vessels, skin) may be prepared from individuals atrisk for Alzheimer's disease, affected by the disease, or not. Tissuemay be obtained as biopsy or autopsy material; cells of interest may beisolated therefrom and then cultured. Also provided are extracts ofcells (e.g., cytoplasm, membrane); at least partially purified nucleicacid and protein therefrom; and methods for their isolation. Thesereagents can be used to establish detection limits for assays, absoluteamounts of gene expression that are indicative of disease or not, ratiosof gene expression that are indicative of disease or not, and thesignificance of differences in such values. These values for positiveand/or negative controls can be measured at the time of assay, before anassay, after an assay, or any combination thereof. Values may berecorded on storage medium and manipulated with computer software;storage in a database allows retrospective or prospective study. Forexample, the database may be physically stored on a tangible media likenote paper or plastic transparency, mechanical switch or electronicvalve, iron core, semiconductor RAM or ROM, magnetic or optical disk, orpaper or magnetic tape. The medium may be erased, refreshed (e.g.,dynamic), or permanent (e.g., static); it may be fixed or transportable.Information may be displayed or projected on a screen (e.g., tangiblemedia such as a cathode ray tube, light emitting diode array, liquidcrystal display). Genes that are increased, decreased, or notsignificantly changed in BEC are identified and related to the alteredcell biology in Alzheimer's disease, and compared with the proprietarygene array data base, the cell biology data base, and the neuropathologyand clinical data bases.

The reliability of diagnosis methods may be improved by (1) decreasingthe incidence of false positive and false negatives and (2) increasingthe sensitivity of detection. For example, the number of different genesthat have a measurable difference in expression (i.e., increased ordecreased) including a subset of disease-specific genes may be at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120,140, 160, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, orintermediate ranges thereof. The amount of change that is consideredsignificant may be at least about 1.5-fold, 2-fold, 2.5-fold, 3-fold,3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold,10-fold, 12-fold, 14-fold, 16-fold, 18-fold, 20-fold, or intermediateranges thereof depending on Bayesian analysis. When expression is barelyor even not detectable, the calculated ratio may be high and is notnecessarily meaningful. The assay is quantitative in the sense thatthere is a direct and measurable relationship between the detectedsignal and gene expression (e.g., the number of transcripts orproteins), but the relationship does not necessarily need to be linear.In addition, a subset of disease-specific genes could be used togenerate diagnostics arrays for AD vascular disorder.

Polynucleotides representative of genes that are increased or decreasedin Alzheimer's disease may be used to identify, isolate, or detectcomplementary polynucleotides by binding assays. Similarly, polypeptidesrepresentative of the gene products that are increased or decreased inAlzheimer's disease may be used to identify, isolate, or detectinteracting proteins by binding assays. Optionally, bound complexesincluding interacting proteins may be identified, isolated, or detectedindirectly though a specific binding molecule (e.g., antibody, naturalor nonnatural peptide mimetic) for the gene product that is increased ordecreased in Alzheimer's disease. Interacting proteins may also beassociated with or cause Alzheimer's disease. Affinity chromatography ofDNA-binding proteins, electrophoretic mobility shift assay (EMSA), one-or two-hybrid system, membrane protein cross-linking, and screening aphage display library are techniques for identifying, isolating, ordetecting interacting proteins.

Candidate compounds useful for treating Alzheimer's disease may interactwith a representative polynucleotide or polypeptide, and be screened fortheir ability to provide therapy or prophylaxis. These products may beused in assays (e.g., diagnosis) or for treatment; conveniently, theyare packaged as assay kits or in pharmaceutical form. Examples of drugsthat are able to control aberrant AD BEC-mediated angiogenesis and/orprevent premature and/or accelerated apoptosis during AD BEC-mediatedbrain capillary morphogenesis (i.e., to reverse the dysfunctionalvascular phenotype) are presented below.

Assaying Polynucleotides or Polypeptides

Binding of polynucleotides or polypeptides may take place in solution oron a substrate. The assay format may or may not require separation ofbound from not bound. Detectable signals may be direct or indirect,attached to any part of a bound complex, measured competitively,amplified, or any combination thereof. A blocking or washing step may beinterposed to improve sensitivity and/or specificity. Attachment of apolynucleotide or polypeptide, interacting protein, or specific bindingmolecule to a substrate before, after, or during binding results incapture of an unattached species. See U.S. Pat. Nos. 5,143,854 and5,412,087.

Polynucleotide, polypeptide, or specific binding molecule may beattached to a substrate. The substrate may be solid or porous and it maybe formed as a sheet, bead, fiber, tape, tube, or wire. The substratemay be made of cotton, silk, or wool; cellulose, nitrocellulose, nylon,or positively-charged nylon; natural, butyl, silicone, orstyrenebutadiene rubber; agarose or polyacrylamide; crystalline siliconor polymerized organosiloxane; crystalline, amorphous, or impure silica(e.g., quartz) or silicate (e.g., glass); polyacrylonitrile,polycarbonate, polyethylene, polymethyl methacrylate, polymethylpentene,polypropylene, polystyrene, polysulfone, polytetrafluoroethylene,polyvinylidenefluoride, polyvinyl acetate, polyvinyl chloride, orpolyvinyl pyrrolidone; or combinations thereof. Optically-transparentmaterials are preferred so that binding can be monitored and signaltransmitted by light. For example, a bead suspended in solution and atthe end of an optical fiber can be interrogated by a light signal (e.g.,blue, red, or green) sent through the optical fiber when an analyte insolution (e.g., probe conjugated to a blue, red, or green label) bindsto the bead, which is attached to the polynucleotide, polypeptide, orspecific binding molecule.

Such reagents would allow capture of a molecule in solution by specificbinding, and then interaction of the molecule with and immobilization tothe substrate. Monitoring gene expression is facilitated by using anordered substrate array or coded library of multiple substrates.

Polynucleotide, polypeptide, or specific binding molecule may besynthesized in situ by solid-phase chemistry or photolithography todirectly attach the nucleotides or amino acids to the substrate.Attachment of the polynucleotide, polypeptide, or specific bindingmolecule to the substrate may be through a reactive group as, forexample, a carboxy, amino, or hydroxy radical; attachment may also beaccomplished after contact printing, spotting with a pin, pipetting witha pen, or spraying with a nozzle directly onto a substrate.Alternatively, the polynucleotide, polypeptide, or specific bindingmolecule may be reversibly attached to the substrate by interaction of aspecific binding pair (e.g., antibody-digoxygenin/hapten/peptideepitope, biotin-avidin/streptavidin, glutathione S transferase orGST-glutathione, lectin-sugar, maltose binding protein-maltose,polyhistidine-nickel, protein A/G-immunoglobulin); cross-linking may beused if irreversible attachment is desired.

By synthesizing polynucleotide, polypeptide, or specific bindingmolecule in situ or otherwise attaching it to a substrate at apredetermined, discrete position or to a coded substrate, an interactingpolynucleotide, polypeptide, or specific binding molecule can beidentified without determining its sequence. For example, apolynucleotide, polypeptide, or specific binding molecule of knownsequence can be determined by its position (e.g., rectilinear or polarcoordinates) or decoding its signal (e.g., combinatorial tag,electromagnetic radiation) on the substrate. A nucleotide or amino acidsequence will be correlated with each position on or decoded signal ofthe substrate. A substrate may have a pattern of differentpolynucleotides, polypeptides, and/or specific binding molecules (e.g.,at least 5, 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350,400, 450, 500, 1000, 2000, 3000, 4000, 5000, 7500, 10,000, 50,000,100,000 or 1,000,000 distinguishable positions) at low or high density(e.g., at least 1,000, 10,000, 100,000 or 1,000,000 distinguishablepositions per cm²). The number of molecules that can be differentiatedby the signal is only limited by factors such as the scale of thereaction; the number and complexity of combinations; interference with aproperty of electromagnetic radiation like wavelength, frequency,energy, polarization; etc.

Multiplex analysis may be used to monitor expression of different genesat the same time in parallel. Such multiplex analysis may be performedusing different polynucleotides, polypeptides, or specific bindingmolecules arranged in high density on a substrate. Simultaneous solutionmethods such as multiprobe ribonuclease protection assay or multiprimerpair amplification associate each transcript with a different length ofdetected product which is resolved by separation on the basis ofmolecular weight. Multiplex analysis may include custom-made diagnosticsarrays for vascular disorder in AD, and could be compared with theproprietary Socratech data bases.

Changes in gene expression may be manifested in the cell by affectingtranscriptional initiation, transcript stability, translation oftranscript into protein product, protein stability, or combinationsthereof. The gene, transcript, or polypeptide can be assayed bytechniques such as in vitro transcription, in vitro translation,Northern hybridization, nucleic acid hybridization, reversetranscription-polymerase chain reaction (RT-PCR), run-on transcription,Southern hybridization, cell surface protein labeling, metabolic proteinlabeling, antibody binding, immunoprecipitation (IP), enzyme linkedimmunosorbent assay (ELISA), electrophoretic mobility shift.assay(EMSA), radioimmunoassay (RIA), fluorescent or histochemical staining,microscopy and digital image analysis, and fluorescence activated cellanalysis or sorting (FACS).

A reporter or selectable marker gene whose protein product is easilyassayed may be used for convenient detection. Reporter genes include,for example, alkaline phosphatase, β-galactosidase (LacZ),chloramphenicol acetyltransferase (CAT), β-glucoronidase (GUS),bacterial/insect/marine invertebrate luciferases (LUC), green and redfluorescent proteins (GFP and RFP, respectively), horseradish peroxidase(HRP), β-lactamase, and derivatives thereof (e.g., blue EBFP, cyan ECFP,yellow-green EYFP, destabilized GFP variants, stabilized GFP variants,or fusion variants sold as LIVING COLORS fluorescent proteins byClontech). Reporter genes would use cognate substrates that arepreferably assayed by a chromogen, fluorescent, or luminescent signal.Alternatively, assay product may be tagged with a heterologous epitope(e.g., FLAG, MYC, SV40 T antigen, glutathione transferase,hexahistidine, maltose binding protein) for which cognate antibodies oraffinity resins are available.

A polynucleotide may be ligated to a linker oligonucleotide orconjugated to one member of a specific binding pair (e.g.,antibody-digoxygenin/hapten/peptide epitope, biotin-avidin/streptavidin,glutathione S transferase or GST-glutathione, lectin-sugar, maltosebinding protein-maltose, polyhistidine-nickel, proteinA/G-immunoglobulin). The polynucleotide may be conjugated by ligation ofa nucleotide sequence encoding the binding member. A polypeptide may bejoined to one member of the specific binding pair by producing thefusion encoded such a ligated or conjugated polynucleotide or,alternatively, by direct chemical linkage to a reactive moiety on thebinding member by chemical cross-linking. Such polynucleotides andpolypeptides may be used as an affinity reagent to identify, to isolate,and to detect interactions that involve specific binding of a transcriptor protein product of the expression vector. Before or after affinitybinding of the transcript or protein product, the member attached to thepolynucleotide or polypeptide may be bound to its cognate bindingmember. This can produce a complex in solution or immobilized to asupport. A protease recognition site (e.g., for enterokinase, Factor Xa,ICE, secretases, thrombin) may be included between adjoining domains topermit site specific proteolysis that separates those domains and/orinactivates protein activity.

Construction of Expression Vector

An expression vector is a recombinant polynucleotide that is in chemicalform either a deoxyribonucleic acid (DNA) and/or a ribonucleic acid(RNA). The physical form of the expression vector may also vary instrandedness (e.g., single-stranded or double-stranded) and topology(e.g., linear or circular). The expression vector is preferably adouble-stranded deoxyribonucleic acid (dsDNA) or is converted into adsDNA after introduction into a cell (e.g., insertion of a retrovirusinto a host genome as a provirus). The expression vector may include oneor more regions from a mammalian gene expressed in the vascular system,especially endothelial cells (e.g., ICAM-2, tie), or a virus (e.g.,adenovirus, adeno-associated virus, cytomegalovirus, fowlpox virus,herpes simplex virus, lentivirus, Moloney leukemia virus, mouse mammarytumor virus, Rous sarcoma virus, SV40 virus, vaccinia virus), as well asregions suitable for genetic manipulation (e.g., selectable marker,linker with multiple recognition sites for restriction endonucleases,promoter for in vitro transcription, primer annealing sites for in vitroreplication). The expression vector may be associated with proteins andother nucleic acids in a carrier (e.g., packaged in a viral particle) orcondensed with chemicals (e.g., cationic polymers) to target entry intoa cell or tissue.

The expression vector further comprises a regulatory region for geneexpression (e.g., promoter, enhancer, silencer, splice donor andacceptor sites, polyadenylation signal, cellular localization sequence).Transcription can be regulated by tetracyline or dimerized macrolides.The expression vector may be further comprised of one or more splicedonor and acceptor sites within an expressed region; Kozak consensussequence upstream of an expressed region for initiation of translation;and downstream of an expressed region, multiple stop codons in the threeforward reading frames to ensure termination of translation, one or moremRNA degradation signals, a termination of transcription signal, apolyadenylation signal, and a 3′ cleavage signal. For expressed regionsthat do not contain an intron (e.g., a coding region from a cDNA), apair of splice donor and acceptor sites may or may not be preferred. Itwould be useful, however, to include mRNA degradation signal(s) if it isdesired to express one or more of the downstream regions only under theinducing condition. An origin of replication may also be included thatallows replication of the expression vector integrated in the hostgenome or as an autonomously replicating episome. Centromere andtelomere sequences can also be included for the purposes of chromosomalsegregation and protecting chromosomal ends from shortening,respectively. Random or targeted integration into the host genome ismore likely to ensure maintenance of the expression vector but episomescould be maintained by selective pressure or, alternatively, may bepreferred for those applications in which the expression vector ispresent only transiently.

An expressed region may be derived from any gene of interest, and beprovided in either orientation with respect to the promoter; theexpressed region in the antisense orientation will be useful for makingcRNA and antisense polynucleotide. The gene may be derived from the hostcell or organism, from the same species thereof, or designed de novo;but it is preferably of archael, bacterial, fungal, plant, or animalorigin. The gene may have a physiological function of one or morenonexclusive classes: adhesion proteins; cytokines, hormones, and otherregulators of cell growth, mitosis, meiosis, apoptosis, senescence,differentiation, or development; soluble or membrane receptors for suchfactors; adhesion molecules; cell-surface receptors and ligands thereof;cytoskeletal and extracellular matrix proteins; cluster differentiation(CD) antigens, antibody and T-cell antigen receptor chains,histocompatibility antigens, and other factors mediating specificrecognition in immunity; chemokines, receptors thereof, and otherfactors involved in inflammation; enzymes producing lipid mediators ofinflammation and regulators thereof; clotting and complement factors;ion channels and pumps; transporters and binding proteins;neurotransmitters, neurotrophic factors, and receptors thereof; cellcycle regulators, oncogenes, and tumor suppressors; other transducers orcomponents of signaling pathways; proteases and inhibitors thereof;catabolic or metabolic enzymes, and regulators thereof. Some genesproduce alternative transcripts, encode subunits that are assembled ashomopolymers or heteropolymers, or produce propeptides that areactivated by protease cleavage. The expressed region may encode atranslational fusion; open reading frames of the regions encoding apolypeptide and at least one heterologous domain may be ligated inregister. If a reporter or selectable marker is used as the heterologousdomain, then expression of the fusion protein may be readily assayed orlocalized. The heterologous domain may be an affinity or epitope tag.

One or more genes involved in abnormal responses of BEC to aniogenicsignaling, aberrant angiogenesis and/or cellular senescence of BEC(e.g., normal or defective) may be expressed or their expressioninhibited by the above.

Screening of Candidate Compounds

Another aspect of the invention are chemical or genetic compounds,derivatives thereof, and compositions including same that are effectivein treatment of Alzheimer's disease and individuals at risk thereof. Theamount that is administered to an individual in need of therapy orprophylaxis, its formulation, and the timing and route of delivery iseffective to reduce the number or severity of symptoms, to slow or limitprogression of symptoms, to inhibit expression of one or more genes thatare transcribed at a higher level in Alzheimer's disease, to activateexpression of one or more genes that are transcribed at a lower level inAlzheimer's disease, or any combination thereof. The efficacy of acandidate compound can be determined by comparing its effects on asubset of disease specific genes in a BEC gene expression data base andon altered AD BEC cellular responses (i.e., phenotype). Determination ofsuch amounts, formulations, and timing and route of drug delivery iswithin the skill of persons conducting in vitro assays, in vivo studiesof animal models, and human clinical trials.

A screening method may comprise administering a candidate compound to anorganism or incubating a candidate compound with a cell, and thendetermining whether or not gene expression is modulated. Such modulationmay be an increase or decrease in activity that partially or fullycompensates for a change that is associated with or may causeAlzheimer's disease. Gene expression may be increased at the level ofrate of transcriptional initiation, rate of transcriptional elongation,stability of transcript, translation of transcript, rate oftranslational initiation, rate of translational elongation, stability ofprotein, rate of protein folding, proportion of protein in activeconformation, functional efficiency of protein (e.g., activation orrepression of transcription), or combinations thereof. See, for example,U.S. Pat. Nos. 5,071,773 and 5,262,300. High-throughput screening assaysare possible (e.g., by using parallel processing and/or robotics).

The screening method may comprise incubating a candidate compound with acell containing a reporter construct, the reporter construct comprisingtranscription regulatory region covalently linked in a cis configurationto a downstream gene encoding an assayable product; and measuringproduction of the assayable product. A candidate compound whichincreases production of the assayable product would be identified as anagent which activates gene expression while a candidate compound whichdecreases production of the assayable product would be identified as anagent which inhibits gene expression. See, for example, U.S. Pat. Nos.5,849,493 and 5,863,733.

The screening method may comprise measuring in vitro transcription froma reporter construct in the presence or absence of a candidate compound(the reporter construct comprising a transcription regulatory region)and then determining whether transcription is altered by the presence ofthe candidate compound. In vitro transcription may be assayed using acell-free extract, partially purified fractions of the cell, purifiedtranscription factors or RNA polymerase, or combinations thereof. See,for example, U.S. Pat. Nos. 5,453,362; 5,534,410; 5,563,036; 5,637,686;5,708,158; and 5,710,025.

Techniques for measuring transcriptional or translational activity invivo are known in the art. For example, a nuclear run-on assay may beemployed to measure transcription of a reporter gene. Translation of thereporter gene may be measured by determining the activity of thetranslation product. The activity of a reporter gene can be measured bydetermining one or more of transcription of polynucleotide product(e.g., RT-PCR of GFP transcripts), translation of polypeptide product(e.g., immunoassay of GFP protein), and enzymatic activity of thereporter protein per se (e.g., fluorescence of GFP or energy transferthereof).

A compound may be screened for its effect on angiogenesis and/orcellular senescence (e.g., normal or defective) in accordance with theabove.

Genetic Compounds for Treatment

Gene activation may be achieved by inducing an expression vector with adownstream region related to a gene which is down regulated inAlzheimer's disease (e.g., the full-length coding region or functionalportions of the gene; hypermorphic mutants, homologs, orthologs, orparalogs thereof) or unrelated to the gene that acts to relievesuppression of gene activation (e.g., at least partially inhibitingexpression of a negative regulator of the gene). Over expression oftranscription or translation, as well as over expressing proteinfunction, is a more direct approach to gene activation. Alternatively,the downstream expressed region may direct homologous recombination intoa locus in the genome and thereby replace or supplement an endogenoustranscriptional regulatory region of the gene with an expressioncassette.

An expression vector may be introduced into a host mammalian cell ortissue, or nonhuman mammal by a transfection or transgenesis techniqueusing, for example, one or more chemicals (e.g., calcium phosphate,DEAE-dextran, lipids, polymers), biolistics, electroporation, naked DNAtechnology, microinjection, or viral infection. Osmotic shock orsurgical procedures may also be used for transfer across the BBB tostimulate transport of vectors into BEC at the abluminal BBB site or atthe luminal site. The introduced expression vector may integrate intothe host genome of the mammalian cell or nonhuman mammal, or bemaintained as an episome. Many neutral and charged lipids, sterols, andother phospholipids to make lipid carriers are known. For example,neutral lipids are dioleoyl phosphatidylcholine (DOPC) and dioleoylphosphatidyl ethanolamine (DOPE); an anionic lipid is dioleoylphosphatidyl serine (DOPS); cationic lipids are dioleoyl trimethylammonium propane (DOTAP), dioctadecyidiamidoglycyl spermine (DOGS),dioleoyl trimethyl ammonium (DOTMA), and1,3-di-oleoyloxy-2-(6-carboxyspermyl)-propylamide tetraacetate (DOSPER).Dipalmitoyl phosphatidylcholine (DPPC) can be incorporated to improvethe efficacy and/or stability of delivery. Proprietary lipidformulations include: FUGENE 6, LIPOFECTAMINE, LIPOFECTIN, DMRIE-C,TRANSFECTAM, CELLFECTIN, PFX-1, PFX-2, PFX-3, PFX4, PFX-5, PFX-6, PFX-7,PFX-8, TRANSFAST, TFX-10, TFX-20, TFX-50, and LIPOTAXI. The polymer maybe cationic dendrimers, polyamides, polyamidoamines, polyethylene orpolypropylene glycols (PEG), polyethylenimines (PEI), polylysines, orcombinations thereof; alternatively, polymeric materials can be formedinto nanoparticles or microparticles. In naked DNA technology, theexpression vector (usually as a plasmid) is delivered to a cell ortissue, where it may or may not become integrated into the host genome,without using chemical transfecting agents (e.g., lipids, polymers) tocondense the expression vector prior to its introduction into the cellor tissue.

A mammalian cell may be transfected with an expression vector; alsoprovided are transgenic nonhuman mammals made by inserting a constructinto the nucleus at a random or targeted location, or as an episome. Inthe previously discussed alternative, a homologous region from a genecan be used to direct integration to a particular genetic locus in thehost genome and thereby regulate expression of the gene at that locus orectopic copies of the gene may be inserted. For example, a knock-outmutation would eliminate gene function and a knock-in mutation wouldreplace the host sequence with a nucleotide sequence of the mutantconstruct (e.g., neomorphic, hypomorphic, hypermorphic). Polypeptide maybe produced in vitro by culturing transfected cells, in vivo bytransgenesis, or ex vivo by introducing an expression vector intoallogeneic, autologous, histocompatible, or xenogeneic cells and thentransplanting the transfected cells (e.g., totipotent or pluripotentstem cell) into a host organism. Special harvesting and culturingprotocols will be needed for transfection and subsequent transplantationof host stem cells into a host mammal. Immunosuppression of the hostmammal post-transplant or encapsulation of the host cells may benecessary to prevent rejection.

The expression vector may be used to replace function of a gene that isdown regulated or totally defective, supplement function of a partiallydefective gene, or compete with activity of the gene. Thus, the cognategene activity of the host may be neomorphic, hypomorphic, hypermorphic,or normal. Replacement or supplementation of function can beaccomplished by the methods discussed above, and transfected mammaliancells or transgenic nonhuman mammals may be selected for high or lowexpression (e.g., assessing amount of transcribed or translated produce,or physiological function of either product) of the downstream region.But competition between the expressed downstream region and aneomorphic, hypermorphic, or normal gene may be more difficult toachieve unless the encoded polypeptides are multiple subunits that forminto a polymeric protein complex. Alternatively, a negative regulator ora single-chain antibody that inhibits function intracellularly may beencoded by the downstream region of the expression vector. Therefore, atleast partial inhibition of genes that are up regulated in MBEC ofAlzheimer's disease may use antisense, ribozyme, RNAi, or triple helixtechnology in which the expression vector contains a downstream regioncorresponding to the unmodified antisense molecule, ribozyme, siRNAduplex, or triple helix molecule, respectively.

Antisense polynucleotides were initially.believed to directly blocktranslation by hybridizing to mRNA but may involve degradation of suchtranscripts of a gene. The antisense molecule may be recombinantly madeusing at least one functional portion of a gene in the antisenseorientation as a downstream expressed region in an expression vector.Chemically modified bases or linkages may be used to stabilize theantisense polynucleotide by reducing degradation or increasing half-lifein the body (e.g., methyl phosphonates, phosphorothioate, peptidenucleic acids). The sequence of the antisense molecule may becomplementary to the translation initiation site (e.g., between −10 and+10 of the target's nucleotide sequence).

Ribozymes catalyze specific cleavage of an RNA transcript or genome. Themechanism of action involves sequence-specific hybridization tocomplementary cellular or viral RNA, followed by endonucleolyticcleavage. It may or may not be dependent on ribonuclease H activity. Theribozyme includes one or more sequences complementary to the subject RNAas well as catalytic sequences responsible for RNA cleavage (e.g.,hammerhead, hairpin, axehead motifs). For example, potential ribozymecleavage sites within a subject RNA are initially identified by scanningthe subject RNA for ribozyme cleavage sites which include the followingtrinucleotide sequences: GUA, GUU and GUC. Once identified, anoligonucleotide of between about 15 and about 20 ribonucleotidescorresponding to the region of the subject RNA containing the cleavagesite can be evaluated for predicted structural features, such assecondary structure, that can render candidate oligonucleotide sequencesunsuitable. The suitability of candidate sequences can then be evaluatedby their ability to hybridize and cleave target RNA.

siRNA refers to double-stranded RNA of at least 20-25 basepairs whichmediates RNA interference (RNAi). Duplex siRNA corresponding to a targetRNA may be formed by separate transcription of the strands, coupledtranscription from a pair of promoters with opposing polarities, orannealing of a single RNA strand having an at least partiallyself-complementary sequence. Alternatively, duplexedoligoribonucleotides of at least about 21 to about 23 basepairs may bechemically synthesized (e.g., a duplex of 21 ribonucleotides with 3′overhangs of two ribonucleotides) with some substitutions by modifiedbases being tolerated. Mismatches in the center of the siRNA sequence,however, abolishes interference. The region targeted by RNA interferenceshould be transcribed, preferably as a coding region of the gene.Interference appears to be dependent on cellular factors (e.g.,ribonuclease III) that cleave target RNA at sites 21 to 23 bases apart;the position of the cleavage site appears to be defined by the 5′ end ofthe guide siRNA rather than its 3′ end. Priming by a small amount ofsiRNA may trigger interference after amplification by an RNA-dependentRNA polymerase.

Molecules used in triplex helix formation for inhibiting expression of agene that is up regulated should be single-stranded and composed ofdeoxyribonucleotides. The base composition of these oligonucleotidesmust be designed to promote triple helix formation by Hoogsteen basepairing rules, which generally require sizeable stretches of eitherpurines or pyrimidines to be present on one strand of the duplex.Nucleotide sequences can be pyrimidine-based and result in TAT and CGCtriplets across the three associated strands. The pyrimidine-richmolecules provide base complementarity to a purine-rich region of asingle strand of the duplex in a parallel orientation to that strand. Inaddition, triple helix forming molecules can be chosen that arepurine-rich (e.g., containing a stretch of guanines). These moleculesmay form a triple helix with a DNA duplex that is rich in GC pairs, inwhich the majority of the purines are located on a single strand of thetargeted duplex, resulting in GGC triplets across the three strands inthe triplex.

Antibody specific for a gene product increased in Alzheimer's diseasecan be used for inhibition or detection. Polyclonal or monoclonalantibodies may be prepared by immunizing animals (e.g., chicken,hamster, mouse, rat, rabbit, goat, horse) with antigen, and optionallyaffinity purified against the same or a related antigen. Antibodyfragments may be prepared by proteolytic cleavage or geneticengineering; humanized antibody and single-chain antibody may beprepared by transplanting sequences from the antigen binding domains ofantibodies to framework molecules. In general, other specific bindingmolecules may be prepared by screening a combinatorial library for amember which specifically binds antigen (e.g., phage display library).Antigen may be a full-length protein encoded by the gene or fragment(s)thereof. See, for example, U.S. Pat. Nos. 5,403,484; 5,723,286;5,733,743; 5,747,334; and 5,871,974.

Genes involved in abnormal responses to angiogenic signaling, aberrantbrain capillary morphogenesis and BEC differentiation and/or cellularsenescence (e.g., normal or defective) may be expressed or theirexpression inhibited by the above.

Formulation of Compositions

Compounds of the invention or derivatives thereof may be used as amedicament or used to formulate a pharmaceutical composition with one ormore of the utilities disclosed herein. They may be administered invitro to cells in culture, in vivo to cells in the body, or ex vivo tocells outside of an individual that may later be returned to the body ofthe same individual or another. Such cells may be disaggregated orprovided as solid tissue. Examples of drugs that prevent aberrant ADBEC-mediated or SIPS BEC-mediated brain capillary tube formation duringin vitro assays (i.e., reverse the dysfunctional vascular phenotype) arepresented below.

Compounds or derivatives thereof may be used to produce a medicament orother pharmaceutical compositions. Use of.compositions which furthercomprise a pharmaceutically acceptable carrier and compositions whichfurther comprise components useful for delivering the composition to anindividual are known in the art. Addition of such carriers and othercomponents to the composition of the invention is well within the levelof skill in this art.

Pharmaceutical compositions may be administered as a formulation adaptedfor passage through the blood-brain barrier or direct contact with theendothelium. Alternatively, pharmaceutical compositions may be added tothe culture medium. In addition to the active compound, suchcompositions may contain pharmaceutically-acceptable carriers and otheringredients known to facilitate administration and/or enhance uptake(e.g., saline, dimethyl sulfoxide, lipid, polymer, affinity-based cellspecific-targeting systems). The composition may be incorporated in agel, sponge, or other permeable matrix (e.g., formed as pellets or adisk) and placed in proximity to the endothelium for sustained, localrelease. The composition may be administered in a single dose or inmultiple doses which are administered at different times.

Pharmaceutical compositions may be administered by any known route. Byway of example, the composition may be administered by a mucosal,pulmonary, topical, or other localized or systemic route (e.g., enteraland parenteral). The term “parenteral” includes subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intrathecal, andother injection or infusion techniques, without limitation.

Suitable choices in amounts and timing of doses, formulation, and routesof administration can be made with the goals of achieving a favorableresponse in the individual with Alzheimer's disease or at risk thereof(i.e., efficacy), and avoiding undue toxicity or other harm thereto(i.e., safety). Therefore, “effective” refers to such choices thatinvolve routine manipulation of conditions to achieve a desired effect.

A bolus of the formulation administered to an individual over a shorttime once a day is a convenient dosing schedule. Alternatively, theeffective daily dose may be divided into multiple doses for purposes ofadministration, for example, two to twelve doses per day. Dosage levelsof active ingredients in a pharmaceutical composition can also be variedso as to achieve a transient or sustained concentration of the compoundor derivative thereof in an individual, especially in and aroundvascular endothelium of the brain, and to result in the desiredtherapeutic response or protection. But it is also within the skill ofthe art to start doses at levels lower than required to achieve thedesired therapeutic effect and to gradually increase the dosage untilthe desired effect is achieved.

The amount of compound administered is dependent upon factors known to aperson skilled in the art such as bioactivity and bioavailability of thecompound (e.g., half-life in the body, stability, and metabolism);chemical properties of the compound (e.g., molecular weight,hydrophobicity, and solubility); route and scheduling of administration,and the like. For systemic administration, passage of the compound orits metabolite through the blood-brain barrier is important. It willalso be understood that the specific dose level to be achieved for anyparticular individual may depend on a variety of factors, including age,gender, health, medical history, weight, combination with one or moreother drugs, and severity of disease.

The term “treatment” of Alzheimer's disease refers to, inter alia,reducing or alleviating one or more symptoms in an individual,preventing one or more symptoms from worsening or progressing, promotingrecovery or improving prognosis, and/or preventing disease in anindividual who is free therefrom as well as slowing or reducingprogression of existing disease. For a given individual, improvement ina symptom, its worsening, regression, or progression may be determinedby an objective or subjective measure. Efficacy of treatment may bemeasured as an improvement in morbidity or mortality (e.g., lengtheningof survival curve for a selected population). Prophylactic methods(e.g., preventing or reducing the incidence of relapse) are alsoconsidered treatment. Treatment may also involve combination with otherexisting modes of treatment (e.g., ARICEPT or donepezil, COGNEX ortacrine, EXELON or rivastigmine, REMINYL or galantamine, antiamyloidvaccine, Aβ-lowering therapies, mental exercise or stimulation; see forreview Zlokovic, Adv. Drug Deliv. Rev. 54:1533-1660, 2002). Thus,combination treatment with one or more other drugs and one or more othermedical procedures may be practiced.

Similarly, diagnosis according to the invention may be practiced withother diagnostic procedures. For example, endothelium of the vascularsystem, brain, or spinal cord (e.g., blood or leptomeningeal vessels)may be assayed for a change in gene expression profiles usingdisease-specific molecular diagnostics kits (e.g., custom made arrays,multiplex QPCR, multiplex proteomic arrays). In addition, a noninvasivediagnostic procedure (e.g., CAT, MRI, SPECT, or PET) may be used incombination to improve the accuracy and/or sensitivity of diagnosis.Early and reliable diagnosis is especially useful to for treatments thatare only effective for mild to moderate Alzheimer's disease or onlydelay its progression.

The amount which is administered to an individual is preferably anamount that does not induce toxic effects which outweigh the advantageswhich result from its administration. Further objectives are to reducein number, diminish in severity, and/or otherwise relieve suffering fromthe symptoms of the disease in the individual in comparison torecognized standards of care. The invention may also be effectiveagainst neurodegenerative disorders or cognitive impairment in general:for example, dementia, depression, confusion, Creutzfeldt-Jakob or madcow disease, Huntington's disease, loss of motor coordination, multiplesclerosis, Parkinson's disease, Pick disease and other brain storagedisorders (e.g., amyloidosis, gangliosidosis, lipid storage disorders,mucopolysaccharidosis), stroke, syncope, and vascular dementia. Thus,treatment may be directed at an individual who is affected or unaffectedby the neurodegenerative disease; it may improve cognitive function. Theefficacy of treatment may be determined by monitoring cerebral bloodflow (CBF) and/or blood-brain barrier (BBB) function.

Production of compounds according to present regulations will beregulated for good laboratory practices (GLP) and good manufacturingpractices (GMP) by governmental agencies (e.g., U.S. Food and DrugAdministration). This requires accurate and complete record keeping, aswell as monitoring of QA/QC. Oversight of patient protocols by agenciesand institutional panels is also envisioned to ensure that informedconsent is obtained; safety, bioactivity, appropriate dosage, andefficacy of products are studied in phases; results are statisticallysignificant; and ethical guidelines are followed. Similar oversight ofprotocols using animal models, as well as the use of toxic chemicals,and compliance with regulations is required.

The following examples substantiate the claims, inter alia, that thereis dysregulation of vascular function in Alzheimer's disease: e.g.,abnormal responses to angiogenic signaling, apoptosis, aniokis, and/ormitotic catastrophe during brain capillary morphogenesis, aberrantcapillary formation throughout all stages (stage I—vaculolization, stageII—tube formation, stage III—network formation, stage IV—remodeling),cellular senescence, presence of nonfunctional capillaries, and loss ofBBB and CBF regulatory functions, which are all related to changes inexpression of disease-specific genes. This can be used as a prognosticindication for diagnosis and treatment. But they are merely illustrativeof the invention, and are not intended to restrict or otherwise limitits practice.

EXAMPLES

Human Subjects

Microvascular brain endothelial cells (BEC) are representative of thesite of the BBB. They were cultured from human brain tissue (Brodmann'sareas 9 and 10) obtained at autopsy with the postmortem interval (PMI)typically between 3 hr and 6 hr, or from biopsy during brain surgery forepilepsy or brain trauma. The groups of patients and controls used fordifferent studies are given in Tables 1-8. Each Table contains providesinformation on age, gender, PMI, cause of death, the presence ofvascular risk factors, angiopathy, Braak stage, CERAD stage, and CDR(cognitive dementia rate) score for each studied individual. Total RNAwas isolated from primary cultures of BEC at passages 2-4 (P2 to P4).

In our first gene expression analysis using Affymetrix U95A chips(12,500 genes), we compared six AD patients (Table 1) with sixage-matched controls (Table 2), corrected for normal aging processwith-five young controls (Table 3). The characteristics of studied ADcases vs. age-matched controls were: age, 72.5±3.45 vs. 72.2±6.01(mean±s.e., years), gender ratio female/male (F=0 and M=1) of 0.43 vs.0.50, PMI of 3.80±0.41 vs. 4.51±0.75 (mean±s.e., hr), cause of death inboth groups were similar (e.g., cardiac arrest, respiratory failure),incidence of vascular risk factors 4/6 vs. 4/6 was similar, the presenceof amyloid angiopathy were 6/6 vs. 2/6, Braak stage V-VI vs. 0-0I, CERADF/M ratio in AD of 0.5 vs. F/M ratio in controls of 0.5, CDR 3.83±0.29(AD cases) vs. 0.08±0.03 (age-matched controls). Characteristics ofyoung controls were: age of 23.4±3.80 (mean±s.e., years), F/M ratio of0.6, PMI of 4.46±0.62 (mean±s.e., hours), cause of death was trauma, novascular risk factors; Braak and CERAD were zero in all cases (not shownin Table 3), and family history did not reveal cognitive problems (CDRwas not determined).

In our second gene expression analysis using Affymetrix U133A and U133Bchips (45,000 genes), 11 AD patients (Table 4) were compared with fiveage-matched controls (Table 5), corrected for normal aging process byfive middle age controls (Table 6), and five young controls (Table 7).The characteristics of AD cases vs. age-matched controls were: age,80.6±3.65 vs. 82.0±3.87 (mean±s.e., years), gender ratio female/male(F=0 and M=1) of 0.45 vs. 0.40, PMI of 3.64±0.26 vs. 3.53±0.96(mean±s.e., hours), cause of death in both groups were similar (e.g.,cardiac arrest, respiratory failure), incidence of vascular risk factorswas similar 8/11 vs. 4/5, the presence of amyloid angiopathy was 9/11vs. 3/5, Braak stage V-VI for AD vs. 0-I-I-II for age-matched, CERAD F/Mratio for AD of 0.72 vs. F/M ratio for age-matched of 0.4, CDR 3.83±0.29(AD) vs. 0.08±0.03 (controls). Characteristics of middle age controlswere: age of 59.4±1.20 (mean±s.e., years), F/M ratio of 0.4, PMI of4.75±0.22 (mean±s.e., hours), cause of death cardiac arrest,respiratory, incidence of vascular risk factors 2/5, angiopathy 0/5,Braak 0, CERAD 0, CDR 0. Characteristics of young controls were: age of27.6±4.67 (mean±s.e., years), F/M ratio 0.4, PMI 4.46±0.62 (mean±s.e.,hours), cause of death was trauma, no vascular risk factors; Braak andCERAD were zero in all cases (not shown in Table 7), and family historydid not reveal cognitive problems (CDR was not determined).

Table 8 illustrate cases used in the senescence study. Thecharacteristics of studied eight AD cases vs. five age-matched controlswere: age, 78.3±3.71 vs. 72.0±7.39 (mean±s.e., years), gender ratiofemale/male (F=0 and M=1) 0.5 vs. 0.6, PMI of 3.85±0.93 vs. 4.50±0.97(mean±s.e., hours), cause of death in both groups were similar, i.e.,cardiac or respiratory arrest (not shown in Table 8), incidence ofvascular risk factors was comparable in both groups between 60 and 70%of cases had a vascular risk factor (not shown in Table 8), the presenceof amyloid angiopathy was 6/8 vs. 0/5, Braak stage V-VI for AD vs. 0-0-Ifor controls, CERAD F/M ratio for AD of 0.62 vs. 0 for controls, CDR inAD close to 4 in controls 0 (not shown in Table 8). Young controls were:age of 19.3.6±3.53 (mean±s.e., years), F/M ratio of 0.7, PMI (biopsycases), no vascular risk factors; no angiopathy, CERAD zero, CDR zero(not shown in Table 8).

Neuropathological Analysis

Tissue blocks (1 cm³) from autopsy cases were fixed in 10%neutral-buffered formalin, pH 7.3 (Sigma), and embedded in paraffin orsnap-frozen in liquid nitrogen-chilled isopentane. The tissue sampleswere obtained from the superior and middle frontal gyrus (Brodmann'sareas 9 and 10). Tissue sections were stained with either hematoxylinand eosin (H&E) stain or thioflavin S by a modified Bielschowsky silverimpregnation method (Gallyas stain). Thioflavin S stained sections wereviewed through a Zeiss fluorescence microscope equipped with a narrowband, blue/violet filter from 400 nm to 455 nm. Two independentobservers performed the examination. Diagnosis of Alzheimer's diseasewas made according to a modified CERAD (Consortium to Establish aRegistry for Alzheimer's Disease) protocol (see Hyman and Trojanowski,J. Neuropathol Exp. Neurol. 56:1095-1097,1997). In most cases, Braakanalysis was performed in parallel.

Isolation and Culture of Human BEC.

BEC were isolated postmortem as reported (Mackic et al., J. Clin.Invest. 102:734-743,1998). Briefly, brain tissue was cut into smallpieces, and then mechanically dissociated using a loose-fitting cellhomogenizer in RPMI 1640 with 2% fetal calf serum (FCS) andpenicillin/streptomycin. The homogenate was then fractionated over 15%dextran by centrifugation at 10,000 g for 10 min to obtain a brainmicrovessel pellet. Microvessels were further digested with 1 mg/ml ofcollagenase/dispase and 5 μl/ml of DNase in FCS-enriched medium for 1 hrat 37° C. Subsequently the cell suspension was centrifuged at 1000 g for5 min, and the cell pellet was plated on fibronectin-coated flasks inRPMI 1640 with 10% FCS, 10% NuSerum, endothelial cell growth factors,nonessential amino acids, vitamins, and penicillin/streptomycin (Mackicet al., J. Clin. Invest. 102:734-743, 1998).

Characterization of BEC

The P0 primary cultures were grown to confluence, and sorted based onLDL binding using the Dil-Ac-LDL method following the manufacturer'sinstructions (Biomedical Technology). Briefly, cells were incubated withDil-Ac-LDL ligand for 4 hr at 37° C., trypsinized, and then separated byfluorescence activated 25 cell sorting (FACS). Labeled and unlabeledhuman umbilical vein endothelial cells (HUVEC) were used to set gatinglimits as positive and negative controls, respectively. Unlabeled MBECwere used to control for possible background staining or differencesbased on cell size. Positively sorted cells were plated on fibronectin-or collagen-coated flasks in the medium described above. Cultures weregrown in 5% CO₂ and split 1:3 at confluency with collagenase/dispase(Mackic et al., J.

Clin. Invest. 102:734-743,1998). Cells were characterized on cytospins(cells centrifuged onto slides) with a panel of cell-specific antibodiesincluding antibodies against Factor VIII or CD105 (endothelium), CD11b(monocyte/microglia), glial fibrillar acidic protein (astrocytes),a-actin (vascular smooth muscle), and neurofilament-α (neurons). Cellswere greater than 98% positive for Factor VIII and CD105, but negativefor the other markers confirming their endothelial origin.

Immunocytochemical Analysis

Air-dried cryostat sections (10 μm) of the frontal cortex adjacent tothe BEC isolation site and cytospins of subconfluent BEC were used forimmunocytochemical analysis. For single staining, after incubation withprimary antibody, preparations were treated with biotinylated secondaryIgG and incubated with avidin-biotin-HRP (Vector Laboratories). Bindingwas detected with an SG peroxidase detection kit (blue/gray; VectorLaboratories). For double labeling, after incubation with the secondprimary antibody, sections were treated with biotinylated IgG, anddetected with NovaRED (Vector Laboratories). Imaging was accomplishedusing an Axiophot II microscope (Carl Zeiss) equipped with SPOT digitalcamera. Some examples of antibodies used for immunocytochemical analysisinclude Aβ₄₀, rabbit anti-human,1:1,000 (1 mg/ml, Chemicon Intl.); Aβ₄₂,rabbit anti-human, 1:1,000 (1 mg/ml); gax, rabbit polyclonal againstC-terminal region of the rat gax protein (amino acids SDHSSEHAHL), 1:500(7 mg/ml, provided by Dr Kenneth Walsh); integrins avβ3 and avβ5(Chemicon Intl.); the mouse monoclonal antibody to the heavy chain ofhuman LRP-1 designated 8G1, which is specific for human LRP-1 andrecognizes an epitope on the 515 kDa subunit, 1:300 (1.5 mg/ml); cyclinB2, goat anti-human polyclonal, 1:100 (0.2 mg/ml, Santa CruzBiotechnology); CD105 (clone SMG), mouse anti-human, 1:100 (0.1 mg/ml,Serotec); Von Willebrand Factor, rabbit anti-human monoclonal, 1:200(5.6 mg/ml, DAKO); GFAP, mouse anti-bovine polyclonal, 1:500 (11.7mg/ml, DAKO); CD11 b, mouse anti-human monoclonal, 1:500 (4.2 mg/ml,DAKO); α-actin, mouse anti-human monoclonal, 1:100 (0.2 mg/ml,Oncogene); plectin, goat anti-human polyclonal, 1:100 (0.2 mg/ml, SantaCruz Biotechnology); AFX-1, rabbit anti-human polyclonal, 1:1,000 (0.8mg/ml, Sigma); tissue transglutaminase TG2, rabbit anti-humanpolyclonal, 1:200 (0.2 mg/ml, Calbiochem); E2F transcription factor,rabbit anti-human polyclonal, 1:1,000 (0.2 mg/ml, Santa CruzBiotechnology); MMP1, goat anti-human polyclonal, 1:1,000 (0.2 mg/ml,Santa Cruz Biotechnology), ankyrin G, mouse anti-human, 1:1,000 (0.2mg/ml, Santa Cruz Biotechnology); p53, mouse anti-human, 1:100 (0.4mg/ml, DAKO); human active caspase-3, 1:250 (1 mg/ml; Promega); and p16,mouse anti-human, 1:50 (0.03 mg/ml, BD Pharmingen).

Capillary Morphogenesis Assays

The 3-D system has been described in detail by Davis et al. (J. CellSci. 114:917-930, 2001). Here, the system was used to assay theresponsiveness of AD BEC and SIPS BEC to angiogenic stimulation.Briefly, 10⁶ MBEC/ml were suspended within 3-D collagen matrices at 25μl per well in the serum-free culture medium 199 containing VEGF (40ng/ml) and bFGF (40 ng/ml) in 5% CO₂ at 37° C. The cells were fixed with3% glutaraldehyde in phosphate buffered saline. The sections werestained with hematoxylin/eosin and Hoechst 33342. The formation ofintracellular vacuoles (stage I), tubules (stage II), and multicellulartubes and networks (stage III) were determined at 4 hr to 16 hr and 24hr, respectively. The number of apoptotic cells were determined between4 hr and 24 hr using double TUNEL/Hoechst staining. At least 200 cellswere evaluated from an individual well. Light output (lumens) wasquantified at 200 X magnification by counting four fields derived fromtriplicate wells.

In the 2-D system, collagen matrigels of matrix composition containing asupplement of growth factors were used according to manufacturer'sinstructions (BD Biosciences, Bedford, Mass.). In the 2-D matrix system,2×10⁴ cells/well were plated.

Drugs Used in Angiogenesis in Vitro Assays

ZVAD-fmk (50 μM, Sigma), p38 MAPK antagonists including SB SB202190 (SB,10 μM), and plasma-derived activated protein C (APC, prepared by Dr. J.H. Griffin's laboratory, 5-100 nM) were used.

RNA Isolation from MBEC

About 5×10⁵ MBEC were plated in a 100 mm tissue culture dish. MBEC werecultured for 3 to 5 days until the monolayer was subconfluent (about80%). Total RNA was isolated using TRIZOL reagent (Life Technologies)according to the manufacturer's instructions: cells were homogenized ina monophasic solution comprised of phenol and guanidine isothiocyanate,add chloroform and separate phases, differentially precipitate RNA, andwash and solubilize RNA (U.S. Pat. No. 5,346,994). Total RNA wasvisualized by gel electrophoresis and analyzed by spectrophotometry toassess the purity and integrity of the preparation.

Preparation of Labeled Target

Total RNA (10 μg) from each sample was used to generate high fidelitycDNA, which was modified at the 3′ end to contain an initiation site forT7 RNA polymerase following the manufacturer's instructions (SUPERCHOICEkit, Life Technologies). Upon completion of cDNA synthesis, 1 μg ofproduct was used in an in vitro transcription (IVT) reaction thatcontained biotinylated UTP and CTP which were labeled for detectionfollowing hybridization to the array following the manufacturer'sinstructions (ENZO). Full-length IVT product (20 μg) was subsequentlyfragmented in 200 mM Tris-actetate (pH 8.1), 500 mM KOAc, and 150 mMMgOAc at 94° C. for 35 min. Following fragmentation, all componentsgenerated throughout the processing procedure (cDNA, full-length cRNA,and fragmented cRNA) were analyzed by gel electrophoresis to assess theappropriate size distribution prior to array hybridization.

High Density Oligonucleotide Array Hybridization

Samples were subjected to gene expression analysis with the AffymetrixU95A chip (12,500 genes) or Affymetrix U133A and U133B chips (45,000genes). All procedures have been performed according to themanufacturer's instructions. The detailed protocol for sample processingof Affymetrix microarrays and documentation of the sensitivity andquantitative aspects of the method can be found in the Affymetrixmanual.

Data Analysis and Comparative Results

Results with selected genes are discussed below. Although GENECHIPtechnology was used here, similar results are expected if another arraytechnology was used such as spotted arrays (Affymetrix) or printedarrays (Rosetta). Moreover, differential display (U.S. Pat. No.5,665,547); serial analysis of gene expression (U.S. Pat. No. 5,866,330,Genyzme); bead arrays analyzed by fiber optics (WO 98/50782, lllumina)or sorting (U.S. Pat. No. 6,265,163, Lynx) are expected to arrive atsimilar results. Similarly, biosensors to detect protein (U.S. Pat. No.6,329,209) or a cell (U.S. Pat. No. 6,210,910) can be used for geneexpression profiling.

Statistical analysis was performed using Bayesian correction forAffymetrix data (Baldi and Long, Bioinformatics 17:509-519, 2001; Longet al., J. Biol. Chem. 276:19937-19944, 2001). The present analysis usedthe following criteria: 2-fold ratio, Bayesian adjustment of a signal at500 (expression), and 0.05 Bayesian p-log.

Quantitative PCR

The same cDNA was used for microarray hybridization and QRT-PCR analysisfor a subset of genes. mRNA quantitation was performed using Taq-Man™chemistry with fluorescently tagged oligonucleotide probes. Fluorescentintensity was detected by the Perkin-Elmer Applied Biosystem SequenceDetector 7700. Data were analyzed using Perkin-Elmer Sequence DetectorSoftware version 1.6.3. Comparative analysis was performed using thedelta-delta Ct approach as described by Applied Biosystems.

Western Blot Analysis

Cell lysates were prepared from subconfluent BEC cultures, BEC atdifferent stages of capillary morphogenesis or BEC exposed to SIPS (seebelow H₂O₂ model of SIPS). Antibodies for Western blot analysis includedcaspase 3, rabbit anti-human polyclonal, 1:100 (0.5 mg/ml, BDPharMingen); p53, mouse anti-human monoclonal, 1:200 (0.1 mg/ml,Oncogene); p16, mouse anti-human monoclonal, 1:250 (0.5 mg/ml, BDParMingen); p21, mouse anti-human, 1:200 (0.4 mg/ml, Oncogene); Rb(Santa Cruz), p27 (Santa Cruz), fibronectin (Santa Cruz), PAI-1 (SantaCruz) or phosphorylated p38 MAPK; gax, rabbit polyclonal againstC-terminal region of the rat gax protein (amino acids SDHSSEHAHL), 1:500(7 mg/ml, provided by Dr Kenneth Walsh); and β-actin, goat anti-humanpolyclonal, 1:2,500 (0.2 mg/ml, Santa Cruz Biotechnology). The secondaryantibody was HRP-conjugated and peroxidase activity was detected withenhanced chemiluminescence detection kit (ECL, Pierce). The relativeabundance of the primary antigen was determined by scanning densitometryusing β-actin as an internal control.

FACS Analyses of Integrins and Annexin V

FACS analysis of integrins avp3 or avp5 was performed using mouseanti-human avp3 or avp5 antibodies (Chemicon Intl.), respectively, andFITC-goat anti-mouse secondary antibody.

Blood Vessel Quantification and Immunocytochemical Analysis

To determine the size and number of vessels in the gray matter offrontal cortex (areas 9 and 10), tissue sections were stained withanti-CD105 (i.e., endoglin), which labels abluminal site of brainendothelium and anti-CD31 (i.e., PECAM) which labels luminal side ofendothelium. Adjacent sections were assessed for amyloid burden withthioflavin S and Gallyas histochemistry, and semiquantifiedimmunohistochemically with anti-Aβ₁₋₄₀ and anti-Aβ₁₋₄₂ staining ofplaques and vascular amyloid. Vessels were imaged from four randomlyselected fields of cortex (200 μm² each) using an AXIOPHOT microscope(Zeiss) equipped with a SPOT digital camera and PHOTOSHOP software ver.5.5 (Adobe). Quantification of vessels was based on external endothelialcross-sectional diameters using IMAGEPRO software. Vessels weresegregated by size as follows: 6-10 μm (capillaries), 10-30 μm(precapillaries and arterioles), or greater than 30 μm (small arteries).

Senescence-Associated-β-Galactosidase (SA-β-gal)

The proportion of brain endothelial cell positive for SA-β-gal activitywas determined as described by Dimri et al. (Proc. Natl. Acad. Sci. USA92:9363-9367, 1995). Subconfluent cultures were fixed with 2%formaldehyde and 0.2% glutaraldehyde. The presence of SA-β-gal activitywas determined by incubation with 1 mg/ml solution of5-bromo-4-chloro-3-indolyl β-D-galatopyranosoide in 40 mM Na citricacid,. 5 mM K₃FeCN6, 5 mM K₄FeCN₆, 150 mM NaCl, 2 mM MgCl₂ diluted inphosphate-buffered saline (pH 6). The cells were rinsed twice withphosphate buffer saline and washed with methanol.

Cell Cycle Analysis

DNA content was determined with bromodeoxyuridine (BrdU) incorporationand propidium iodide staining as described (Giaretti et al., Exp. CellRes. 182:290-295, 1989). The brain endothelial cell cells were stainedwith 50 μg/mL propidium iodide and the S-phase cells were labeled with10 μg/mL BrdU for 1 hr and washed with serum free medium. DNA contentprofiles for BrdU-positive cells are shown. The analysis was based on10,000 cells counted.

TUNEL (Terminal Deoxynucleotidyl Transferase-Mediated in situ endLabeling) Assay

Staining with APO-BRDU kit was performed according to manufacturer'sinstructions (Phoenix Flow Systems).

Nerve Growth Factor (NGF) ELISA

The NGF ELISA (enzyme-linked immunosorbent assay) reagents were fromPromega. The NGF levels were determined in brain endothelial cell cellculture supernatants.

Co-Culture of Endothelial and Neuronal Cells

Endothelial cells were plated on collagen-coated membranes (0.4 μmpores) in the upper chamber of TRANSWELL inserts (Costar) in a dish andcultured in 5% CO₂at 37° C. (Mackic et al., J. Clin. Invest.102:734-743, 1998). For co-cultures, TRANSWELL inserts containingendothelial cells were rinsed with B27/neurobasal medium and transferredto the plate containing one-day old primary rat hippocampal neurons inthe lower chamber. After two days of co-culturing, neurons were fixed in4% paraformaldehyde and the length of neurites determined usingIMAGE-PRO PLUS software (Media Cybernetics).

H₂O₂ Stress-Induced Premature Senescence (SIPS)

Subconfluent BEC cultures (34 days after subculture) were treated withH₂O₂ by adding it to the culture medium for 2 hr. To induce senescence,sublethal doses of H₂O₂ were determined and selected. After treatment,cells were washed with PBS (37° C.) before harvesting, subculturing orincubating with a fresh medium or 3-D collagen gels. H₂O₂-treated cellswere subcultured and analyzed after 1, 2, 3, 4, 5, 6, 24, 48, 72, 96 and120 hr for expression of different proteins involved in the cell cycleregulation and apoptosis. Cells in 60 mm dishes were lysed in SDS samplebuffer directly. For Western blot analysis, proteins were separated bySDS-PAGE and transferred to PVDF membrane followed by incubation withdifferent primary antibodies (see antibodies for.Western blot analysis).SIPS BEC were also prepared for microarray analysis on U95A chips asdescribed above.

Differences in Cell Biology

Preliminary findings have been reported in four abstracts: Chow N, Li F,Brooks A, Zidovetzki R, Hofman F, Zlokovic BV, “Senescence of CerebralEndothelium in Alzheimer's Disease” Soc. Neurosci. Abstract No. 420.9(2002); Hofman F M, Chow N, Kanagala S, Zidovetzki R, Zlokovic B V,“Down Regulation of Gax in Cerebral Endothelium in Alzheimer's Disease”Soc. Neurosci. Abstract No. 328.2 (2002); Kanagala S, Li F, Chow N,Brooks A, Paxhia A, Armstrong D, Zidovetzki R, Hofman F, Zlokovic B V,“Defective Angiogenesis in Alzheimer's Disease” Soc. Neurosci. AbstractNo. 328.2 (2002); and Chow N, Li F, Brooks A, Zidovetzki R, Hofman F,Zlokovic B V, “Cellular senescence of vascular brain endothelium inAlzheimer's disease” Cold Spring Harbor Meeting on Molecular Geneticsand Aging Conference (2002). Preliminary findings will be reported inthree abstracts: Hofman F M, Li F, Chow N, Cheng T, Penn L, Griffin J H,Zlokovic B V “Activated protein C enhances angiogenesis in brainendothelial cells in Alzheimer's disease” Soc. Neurosci. Abstract No.9573 (2003); Chow N, Guo H, Zlokovic B V, “Senescence of cerebralendothelium in Alzheimer's Disease: An in vitro model system” Soc.Neurosci. Abstract No. 3897 (2003); and Sallstrom J F, Brooks A, PaxhiaA, Song X, Guo H, Zlokovic B V, “Refinement of gene expression analysisin Alzheimer's Disease microvascular cells using laser capturemicrodissection” Soc. Neurosci. Abstract No.12271 (2003).

FIGS. 1-2 illustrate in vitro capillary morphogenesis in 3-D collagenmatrices made by primary BEC (P2-P4) derived from six AD patients andsix age-matched controls. The characteristics of patients and controlsare given in Tables 1-2. As shown in FIGS. 1A-1B, BEC differentiation incontrols begins with the formation of intracellular vacuoles (stage I)between 4 hr and 16 hr. No significant apoptosis is observed. Shortlyafter vacuolar stage, BEC elongate to form capillary tubes (FIG. 1C). At24 hr, most BEC differentiate into capillary tubes (stage II; notshown). This model of BEC-mediated capillary morphogenesis in controlsresembles that of systemic endothelial cells (Davis et al., J. Cell.Sci.114, 917-930, 2001), but some differences are discussed below.

In-contrast to control BEC, AD BEC exhibit early apoptotic changes in20%-35% of the population. In the present AD model of brain capillarymorphogenesis, a subpopulation of cells show blebbing of the cytoplasmicmembrane (about 35%), chromatin condensation and/or nuclearfragmentation at 4 hr (FIGS. 1D-1E). Apoptosis of AD BEC could also beobserved at 24 hr during capillary tube formation (FIG. 1F). Ultimately,AD BEC formed poor capillary networks compared to controls.

Counting of cells with vacuoles indicated no significant changes betweenAD and either of the two controls (FIG. 2A). But the number of tubes(FIG. 2B) and the total tube length (FIG. 2C) in the AD model at 24 hrwere 2- to 2.7-fold less than in age-matched or young controls,respectively. This suggests that cells in AD die prematurely before theelongation into capillary tubes.

About 20% of cells in the AD model of brain capillary morphogenesis wereTUNEL positive at 4 hr compared to either young or age-matched controlcultures (FIGS. 3A-3E). To determine a possible mechanism for enhancedapoptosis in AD, Western blot analysis of cell lysates was performed(FIGS. 3F-3H): there was a significant increase in p53 in AD BECcompared to controls at 4 and 12 hr (FIG. 3I) and in caspase 3 in AD BECthroughout the different stages of capillary morphogenesis within 24 hr(FIG. 3J). The greatest increases of both p53 and caspase 3 were foundduring first 4 hr, which suggests a rapid activation of programmed celldeath in AD BEC in response to angiogenic stimulation.

zVAD-fmk, a broad spectrum caspase inhibitor (Faleiro & Lazebnik, J.Cell Biol. 151, 951-959, 2000) was used. The results show that zVAD-fmkprevents apoptosis in AD BEC by preventing the formation of the activeform of caspase 3 (FIG. 4A). Treatment with zVAD has resulted in therestoration of the number of capillary tubes (FIG. 4B) and total tubelength at 24 hr (FIG. 4C) during AD BEC-mediated tubule formation. ZVADalso decreased a number of apoptotic AD BEC by TUNEL staining (notshown).

The morphological changes observed during in vitro angiogenesis assayscorrelated with the reported in situ observation in brains withAlzheimer's disease of atrophic vessels, blebbing of cytoplasmicmembranes, capillary degeneration, tortuous vessels, regions ofdecreased microvascular density, and/or abnormal endothelialproliferation (Miyakawa et al., Virchows Arch. 40:121-129, 1982; de laTorre, Stroke 33:1152-1162, 2002). In contrast to the previous vasculartheory's teaching that brain capillary degeneration is caused by hypoxiadue to cerebral hypoperfusion in Alzheimer's disease (de la Torre,Neurobiol. Aging 21:331-242, 2000), the present findings suggest thatthe microvascular changes observed in brains of individuals withAlzheimer's disease could be due to defective angiogenesis andneovascularization caused by the inability of BEC to differentiate intocapillary tubes, and/or an aberrant response of BEC to growth andangiogenic factors.

To further test whether similar molecular changes preceding apoptosis inour in vitro model also take place in brains in situ, immunodetectionstudies were performed for collagen IV (a vascular basement membranemarker) and p53 (FIGS. 5A-5D) and for collagen IV and caspase-3 (FIGS.5E-H) on brain tissue sections Brodmann's areas 9 and 10 from ADpatients and age-matched controls adjacent to the site of BEC isolationfor in vitro studies (FIGS. 1-4). The in situ staining studies confirmedincreased expression of p53-positive and caspase-3 positive brainmicrovessels in patients with AD compared to age-matched controls, i.e.,about 60% of vessels were positive for both p53 and caspase-3 comparedto less than 20% of positive vessels in controls (FIGS. 5I-5J). Thesestudies are suggestive that p53-dependent apoptosis and caspase-3pro-apoptotic signaling may take place in the part of the vascularsystem in AD brains in situ.

Next, studies with activated protein C (APC) were performed because APChas been recently reported to prevent apoptosis in human brainendothelium through endothelial protein C receptor (EPCR)-dependentactivation of protease activated receptor-1 (PAR-1) by blocking p53 andcaspase-3 pro-apoptotic signaling (Cheng et al., Nature Med. 9:338442,2003). Microarray studies confirmed the presence of both EPCR and PAR-1mRNA in control and AD BEC, and no changes in the level of theirtranscripts in AD BEC vs. control BEC (not shown). FIG. 6A illustratesthat APC (100 nM) significantly improved AD BEC-mediated formation ofcapillary tubes by greater than 50% and that the active catalytic siteserine is required for this effect, which suggests the possibleinvolvement of PARs (FIG. 6B). As a negative control, heat-inactivatedAPC was without effect (FIG. 6B). APC significantly reduced the numberof TUNEL-positive cells and caspase-3 positive cells during ADBEC-mediated brain capillary morphogenesis by 84% and 61%, respectively(FIGS. 6C-6D), which confirms the significant anti-apoptotic activityduring AD BEC-mediated tubule formation. APC (100 nM) also enhancedmigration of AD BEC by>50% (FIG. 6E). This effect was neutralized bymonoclonal C3 anti-APC antibody (Heeb et al., Thromb. Res. 52:3343,1988). EPCR appears to be required for this effect of APC (FIG. 6F).

The pattern of capillary morphogenesis in control BEC is similar toHUVEC (Davis et al., J. Cell Sci. 114:917-930, 2001), but there was asignificant difference in the time course of formation of theintracellular vacuoles: 12 hr to 16 hr for BEC compared to 4 hr to 8 hrfor HUVEC. It is noteworthy that BEC exhibit hairy cytoplasmic processesand their cell bodies become positive for neuronal markers at earlystages of differentiation (i.e., between 1 hr and 4 hr) as during earlystages of neurogenesis (e.g., neuron-specific tubulin TuJ, tyrosinehydroxylase, etc.). This was never seen in other endothelial cellsincluding HUVEC. In addition, a significant number of BEC at this earlystage (1 hr to 4 hr) were able to acquire the morphological appearanceof neurons, showing processes that resemble neuritic or dendriticprocesses, and the shape of cells that resembles the shape of pyramidalcells in the brain, bipolar cells in the retinal ganglion, and/orrounded granular cells from the cerebellum. These findings raise thepossibility that primary BEC may preserve some of the features ofpluripotent precursor cells and could hold eventually a potential todifferentiate towards the neuronal lineage if cultured in theenvironment suitable for neuronal growth and differentiation and in thepresence of neuronal growth factors.

A subpopulation of BEC derived from Alzheimer's disease patients, inaddition to those actively dividing and dying by apoptosis, includesenescent cells. FIG. 7A shows impaired growth of AD BEC compared tocontrol BEC; their population doubling time (PDT) was longer by 2-foldthan for age-matched controls (FIG. 7B). AD BEC compared to control BECat earlier passages express common markers of senescence includingenlarged and flattened morphology and expression ofsenescence-associated-β-galactosidase (SA-β-gal) at pH 6.0 (Campisi etal., Exp. Gerontol. 31:7-12, 1996). There was a progressive increase innumber of SA-β-gal-positive senescent cells in AD BEC from 23 PD (FIG.7C) to 34 PD (FIG. 7D) when most cells become senescent. On the otherhand, AMC BEC cultured under same conditions have insignificant numberof senescent BEC even after 44 PD (FIG. 7E). FIG. 7F shows that AD BECreach a stage of almost complete replicative senescence after about 30cumulative PDs, while AMC BEC at that stage express a negligible numberof senescent cells. The senescent phenotype was further revealed bydeficits in early- and mid-G1 phase in response to serum stimulation inAD BEC (not shown), and increased expression of p16, both at thetranscript and protein level (see below). Thus, it appears thatreplicative senescence of AD BEC in culture reflects senescence of thevascular system in Alzheimer's disease brains, and development ofprematurely-aged dysfunctional vasculature as suggested by molecularanalysis (see below).

To better understand the molecular basis of AD BEC senescence and todevelop a simple model to test different therapeutic strategies for ADvascular disorder, an H₂O₂ model of SIPS (Chen et al., J. Cell Sci. 113:4087-4097, 2000) was adapted to human BEC. BEC treated with sublethalconcentrations of H₂O₂ (300 μM) for 2 hr developed a senescent phenotypewithin 48 hr with all of the phentotypic markers characteristic ofsenescence, including G1 cell arrest (FIGS. 8A-8B), enlarged andflattened morphology, and expression of SA-β-gal at pH 6.0 (FIGS.8C-8D). The concentration of H₂O₂ that induces BEC senescence wasdetermined after initial studies with different concentrations of H₂O₂from 10 μM to 1 mM and by comparing its toxic effect (e.g., LDH release,nuclear condensation, and fragmentation) to its effect on cell cycle andmitosis. These studies revealed that control BEC treated with 300 μMH₂O₂ develop a phenotype indistinguishable from those of senescent BECderived from individuals with Alzheimer's disease. Higher concentrationsof H₂O₂ resulted in BEC apoptosis (not shown).

H₂O₂-treated cells became senescent through a series of molecular eventswhich included changes in the expression of cell cycle regulator genes.The first noticeable early changes were transient p38 phosphorylationand p53 phosphorylation within 10 min and 30 min of exposure to H₂O₂,respectively, followed by induction of cyclin-dependent kinase inhibitor(CDKI) p21^(CIP1) (FIG. 8E) that peaked approximately at 48 hr andremained elevated within next 48 hr to 72 hr (FIG. 8F). Finally, therewas a progressive elevation of CDKI p16^(INK) ^(4a) that stabilized at72 hr (FIG. 8F) and remained elevated for the remaining studied periodof several days (Chow et al., Soc. Neurosci. Abstract No. 420.9, 2002).At 24 hr, there was also lack of Rb phosphorylation. All these changesresulted in withdrawal of brain endothelial cell from the cell cycle,permanent G1 arrest as shown in FIGS. 8A-8B, and development of thesenescent phenotype (FIGS. 8C-8D). H₂O₂-treated cells had higher levelsof fibronectin and PAI-1 (not shown), markers usually associated withsenescence of fibroblasts (Chen et al., J. Cell Sci. 113:4087-4097,2000).

Since p16 has been shown to play a critical role in stabilizingendothelial cell senescent phenotype (Shelton et al., Current Biol.9:939-945,1999; Choi et al., FASEB J. 15:1014-1020, 2001), p16expression during replicative senescence in AD BEC and after SIPS incontrol young BEC was determined by Western blot analysis. FIGS. 9A and9C show a time-dependent progressive increase in p16 expression witheach increasing passage of AD BEC (i.e., shown from passage 5 to passage7). FIGS. 9B and 9D confirm the progressive increase in p16 levels in aH₂O₂ model of BEC SIPS from 0, 3 and 6 days. These findings confirmedthat in both RS AD BEC and SIPS BEC, p16 is critically associated with asenescent phenotype.

The analysis of brain tissue confirmed increased number of p16-positivevascular profiles in AD compared to age-matched controls (FIGS.10A-10F). The number of p16-positive brain vessels in AD was about 40%compared to 5% in controls (FIG. 10G), which suggests that there isvascular senescence in AD brain endothelium in situ. Double stainingwith CD-105 (endoglin), a marker for the abluminal site of theendothelium in situ, and CD31 (PECAM), a marker for the luminal site ofthe endothelium in situ, has revealed flattening of the cell cytoplasmin AD BEC in tissue resembling the senescent changes seen in vitro withthe isolated cells. Loss of cell shape due to loss of stabilizingcytoskeletal elements can make cells prone to mechanical injury and mayobstruct the lumen of microvessels compromising brain circulation, CBFregulation and BBB transport.

H₂O₂-treatment resulted in early transitory phosphorylation of p38 MAPKthat peaked at 1 hr to 2 hr, and returned to normal levels shortly afterthat. It has been proposed that activation of p38 MAPK may controlapoptosis during angiogenesis thus playing a crucial role in vascularremodeling, as for example during FGF-mediated angiogenesis (Mastumotoet al., J. Cell Biol. 156:149-166, 2002). Therefore, the performance ofH₂O₂-treated BEC and RS BEC was assayed for capillary morphogenesis.FIGS. 11A-11C show that both SIPS BEC or RS BEC form an insignificantnumber of tubes compared to untreated normal control cells. Butpreincubation of BEC with SB202190, a p38 MAPK activation inhibitor,significantly improved tube formation in the SIPS model augmenting totaltube length by almost 5-fold (FIG. 11C). Moreover, treatment of earlypassage AD BEC with SB202190 completely restored the number of tubes tothe level of control BEC (FIG. 11D-11F). Thus, an early activation ofp38 MAPK in response to angiogenic signaling in SIPS BEC or AD BEC maylead to massive apoptosis and an imbalance between formation andregression of new vessels in diseases such as Alzheimer's diseasevascular disorder and/or pre-senescent and senescent state of brainendothelial cell.

To further understand molecular events involved in abnormal responses toangiogenic signaling and aberrant angiogenesis in AD BEC and cellularsenescence, we performed several high-throughput screening microarraystudies on BEC (P2-P4) from patients and controls were performed.

In the first analysis (Table 9), changes in gene expression werecompared between six individuals with AD and six age-matched or fiveyoung controls on Affymetrix U95A chips (12,500 genes). The average ageof AD and age-matched controls was about 70 years. There was nosignificant differences in gender ratio, the PMI, and cause of death.The incidence of vascular risk factors was 67% in either group. But ADcases had significantly higher incidence of CAA, i.e., 6/6 (100%) vs.2/6 (33%) than controls. AD cases were Braak stage V-VI compared to 0 or0-I in controls, and CERAD 50/50 moderate to frequent compared tonegative or sparse in controls. The average CDR in this AD group wasclose to 4, while it was zero in age-matched controls. Detailedcharacteristics of the groups are given in Tables 1-3.

In the second analysis (Tables 10-11), changes in gene expression werecompared between 11 individuals with AD and five age-matched, fivemiddle-age, or five young controls on Affymetrix U133A and U133B chips(45,000 genes). The average age of AD and age-matched controls was about80 years. There was no significant differences in gender ratio, PMI, andcause of death. The incidence of vascular risk factors was high andcomparable in both groups, AD (73%) and age-matched controls (80%). Theincidence of CAA was 82% in AD and somewhat lower in age-matchedcontrols (60%). AD cases were all Braak stage V-VI compared to 0-I orI-Il in controls. By CERAD AD were 72/28 frequent to mode-rate, whileage-matched controls were sparse (60%) to moderate (40%). The averageCDR in this AD group was also close to 4, while it was zero inage-matched controls. Detailed characteristics of the groups are givenin Tables 4-8.

In the third analysis (Table 12), the coincidence genes were studiedwith Affymetrix U95A chips between RS-AD BEC vs. early passage AD BEC inthree AD cases compared to SIPS young BEC (four cases) vs. control youngBEC (five cases).

Bayesian correction for data analysis of the Affymetrix U95A, U133A, andU133B array results used the criteria described (i.e., 2-fold ratio,expression 500, 0.05 Bayesian p-log).

The first analysis (Table 9) has revealed significant differences inexpression in a subset of 42 genes (i.e., 0.3% out of 12,500) inAlzheimer's disease compared to age-matched controls (p<0.05 or thedifference is found in greater than 95% of cases). In AD, severaltranscription factors and genes with predicted actions in celldifferentiation and angiogenesis, signal transduction, cytoskeleton,matrix, and cell cycle regulation were significantly dysregulatedcompared to age-matched controls (Table 9). No changes in the expressionof these genes were observed between young and age-matched controls withthe exception of L-3-phopshoserine phosphatase that was significantlyincreased in age-matched vs. young BEC and exhibited further sharpincrease in AD vs. age-matched controls. This suggests that reportedgene expression profile in AD was largely age-independent.

An independent validation of microarray data was performed by QRT-PCRanalysis for a subset of genes including gax, tissue transglutaminase(TG2), eukaryotic translation initiation factor 2 gamma subunit (elF2γ)and asparaginyl tRNA synthetase, a gene that was not altered in AD vs.controls by Bayesian analysis (1.007-fold difference; p=0.91). TheQRT-PCR analysis confirmed the same direction of change for gax, TG2 andelF2γ, and the magnitude of change was even more significant than by themicroarray analysis, i.e., by −16.7-fold, +14.8-fold and −10.1-fold,respectively, and no change in the expression of asparaginyl tRNAsynthetase.

The homeobox gene gax (Gorski & Walsh, Circ. Res. 87:865-872, 2000) wasdown regulated 2-fold in AD BEC. Down regulation of gax mRNA and gaxhomeoprotein was confirmed by QRT-PCR analysis (−16.7-fold) and Westernblot analysis of cell lysates (−3.7-fold). It was also undetectable inbrain microvessels in tissue sections from brains of AD patients, butwas expressed in brain microvessels in age-matched and/or young controls(not shown). Consistent with the prediction that down regulation of gaxwould lead to up regulation of αvβ3 and αvβ5 integrins (Witzenbilcher etal, J. Clin. Invest. 104:1469-1480, 1999), it was confirmed by FACSanalysis that BEC of individuals with AD express consistently higherlevels of cell surface αvβ3 and αvβ5 integrins, but not theαvβiintegrins. The migration of AD vs. control BEC in a Boyden chamberindicated that AD BEC migrate by about 2-fold slower than controls.Increased levels of αvβ3 and αvβ5 integrins, but again not the αvβ1integrins, have been confirmed in brain endothelium in tissue sectionsof individuals with AD for the sites adjacent to the site used for BECisolation.

Although some of these genes (Table 9) have functions relevant to cellgrowth and differentiation, apoptosis, and specialized functions of thevasculature their effects on the biology of BEC in relation to aneurodegenerative disorder have not been previously demonstrated. Thealtered BEC physiology is at least in part manifested in dysregulationof the brain vasculature (e.g., abnormal angiogenic. signaling,activation of programmed cell death, aberrant angiogenesis and/orcellular senescence). Here, it is shown that the differences in geneexpression can be related to changes in the cell biology of thevasculature in patients (i.e., Alzheimer's phenotype) and function ofthe BBB. Anatomic and enzymatic components of the BBB are reviewed inMcComb and Zlokovic (Cerebrospinal fluid and the blood-brain interface,In: Textbook of Pediatric Neurosurgery, Philadelphia, Pa.: Saunders,2000).

The functions discussed here for brain endothelial cells may actindependently, additively, or synergistically in Alzheimer's disease:loss of neurotrophic support, reduced detoxification, dysregulation ofcell growth in the microvasculature (e.g., smooth muscle, endothelialcell) leading to nonsense angiogenesis and incompetent capillarymorphogenesis. This defect may also either reduce the longevity of thevascular system in the brain and/or predispose it to SIPS. The presenceof senescent cells in the vascular system can significantly reducenormal physiological functions of the BBB related to molecular exchangebetween blood and brain and may impair the CBF regulations. Thus,dysregulation of growth and senescence of BEC, can be linked topathogenesis of Alzheimer's disease and mechanisms of disease.

These discoveries shift attention in understanding Alzheimer's diseasefrom plaque formation in the neuronal and vascular compartments to themicrovasculature that comprises the blood-brain barrier. The phenotypicdrift and destabilized gene expression profile of endothelium may resultfrom a disease-specific defect primary to the vascular system. This canhave consequences for pathophysiology of the brain vascular systemleading to clinical dementia. It is envisioned that these pathogenicpathways may be coordinated by “master” key genes which regulate one ormore of the pathways. It is also possible that the primary defect iscaused by somatic mutation and/or chromosomal translocation in brainendothelium that inhibits normal responses of BEC to angiogenicsignaling and differentiation into functional capillary tubes. Westernblot analysis of cell lysates, ELISA of cell culture supernatants,and/or immunocytochemical analysis of brain tissue in situ for severalgene products suggest a general and consistent agreement witholigonucleotide array results. Proteomic studies (e.g., quantitative orsemiquantitative Western blotting, ELISA, and immunostaining) haveconfirmed that the changes in gene expression observed as RNAtranscribed for a subset of genes are also detectable at the level oftranslated protein. In general, the direction of the change in geneexpression (i.e., increased or decreased) is the same but the magnitudeof any difference may vary. This may reflect differences in the cellcultures or samples obtained therefrom, regulation at the level ofprotein translation or processing, saturation of the protein translationor protein processing, or the like.

These findings further indicate that correcting defectivedifferentiation of brain endothelial cells into vascular tubes could bea distinct therapeutic target in Alzheimer's disease. Several gene andmolecular candidates underlying these cellular defects are describedbelow. Preventing programmed cell death of brain endothelium which isstimulated by growth factors and/or during an angiogenic response isclearly a distinct vascular therapeutic target in Alzheimer's disease.Examples of drugs that are able to prevent and/or reverse aberrant ADBEC-mediated angiogenesis or angiogenesis from senescent BEC are givenin FIGS. 4, 6 and 11, with zVAD, APC and p38 MAPK inhibitor,respectively.

Here, we describe in greater detail changes in gene expression that arestatistically significant and therefore considered to be present ingreater than 95% of Alzheimer's disease cases compared to age-matchedcontrols in the first analysis (Table 9) of cases listed in Tables 1-3.The reference list is given below.

Based on expression profiling, the role of gax, a transcription factorwhich is involved in angiogenesis, vascular remodeling, and regulationof the cell cycle and cell migration (1), was determined. The gaxhomeoprotein is down regulated in AD brains in situ, which validates thepresent AD BEC model of defective angiogenesis (Hofman et al., Soc.Neurosci, Abstract No. 328.3, 2002). Bearing in mind the importance ofgax-integrin axis (2), the regulation of αvβ3 and αvβ5 integrins in ADBEC and in microvessels of AD brains in situ was analyzed, and theresults confirmed a significantly increased cell surface expression ofthese integrins in AD BEC in vitro by 5-fold and 3-fold, respectively,and in brain vessels in AD brains in situ by 4-fold and 55%,respectively, based on the number of positive vascular profiles. Thesedata again, validate the importance of the gax-integrin axis in our invitro BEC model.

αvβ3 and αvβ5 interins play a critical role in angiogenic signaling,cell migration and proliferation (3). In previously reported studies, upregulation of αvβ3 and αvβ5 integrins in response to gax down regulationwas linked to increased cell migration (2). In the results presentedhere, upregulated integrin expression led to decreased migration. Thissuggests that the gax-integrin interactions may be cell specific, theseinteractions are aberrant in AD BEC, and/or the result reflects theactivation of a compensatory program that attempts to balance anas-of-yet unidentified defect in angiogenesis in the AD cells.

Cell death during brain capillary morphogenesis in AD may result fromincreased levels of p53 as shown for different cell types includingendothelium (4). Since AD cells migrated significantly slower thancontrols, the increased αvβ3 and αvβ5 integrins expression in AD BEC ismost likely functioning aberrantly, either with decreased integrinaffinity or avidity, or abnormal function (e.g., integrin detachmentfunction that may favor cell death in AD by anoikis (3,5). Therelationship between integrin activity and p53 is not well understood,but it has been reported blocking and/or aberrant integrin signaling maytrigger apoptosis by activating p53 (6). A stabilization of p53 at Ser15phosphorylation site of ataxia teleangiectasia mutated kinase inresponse to DNA damage (7) was not found in the present study (notshown). However, DNA damage still may take place in AD BEC as suggestedby up regulation of AP endonuclease XTH2 gene (Table 9), a nuclear andmitochrondrial DNA repair enzyme (8).

The present gene array analysis strengthens the hypothesis that braincapillary degeneration in AD is independent of hypoxia, by demonstratingno changes in hypoxia-inducible genes, e.g., the hypoxia-induciblefactor 1 (HIF-1) gene and its HIF-1α subunit, a transcription factorwhich is induced by decreased cellular oxygen (9). These gene array dataare also confirmed by the Western blot analysis (not shown). Theexpression of VEGF receptors, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR)and tyrosine kinase receptors implicated in angiogenic signaling (e.g.,tie-2, tie-1, FGF receptors) (10,11) was not significantly altered in ADBEC according to the microarray data, and confirmed for some by FACSanalysis (e.g., VEGFR-1, VEGFR-2) and Western blot analysis of celllysates (not shown).

Altered expression of several genes in AD BEC may contribute to impairedsignal transduction. Sharply increased expression ofL-3-phosphoserine-phosphatase (Table 9) can counteract the activity ofserine/threonine kinases and increase cerebrovascular levels of D-serinewhich blocks the synaptic transmission via N-methyl-D-aspartate glycinereceptor (12). Down regulation of calcium, calmodulin regulated3,5-cyclic nucleotide phosphodiesterase will lead to accumulation ofcyclic adenosine mono phosphate (13). Down regulation of the geneencoding REM, a member of subfamily of RAS-related GTPases that includeRad, Gem, and Kir (14), and up regulation of the gene encodingNef-associated factor 1 beta that associates with several cellulartyrosine kinases and serine/threonine kinases (15) can also modifysignal transduction.

Ankyrin G (ANK-3) is significantly down regulated in AD BEC (Table 9).Ankyrins represent a protein family whose members are associated withmembrane proteins and the actin cytoskeleton, and are involved inregulating several cellular processes (16). Integrin-linked kinase(ILK), a multidomain focal adhesion protein that is involved in adhesionof cells to the matrix and signal transduction mediated by integrins,binds with high affinity to PINCH, a focal adhesion protein, via theN-terminal ankyrin repeat domain (16). So although down regulation ofankyrin G might not be specific for the malfunction of ILK in BEC ofindividuals with AD, it may contribute to deficient signal transductionobserved in these cells.

Gene array analysis indicated down regulation of TINUR NGF-B/nur 77 betatype transcription factor, a member of the steroid/thyroid hormonenuclear receptor superfamily (17), which predicts a deficient responseof AD BEC to thyroid/steroid hormones during differentiation. C-mafproto-oncogene, a member of a large family of basic zipper transcriptionfactors (18) is also down regulated in AD. C-maf can form heterodimerswith jun and fos and may induce cell death through its control of p53expression (18). The AFX forkhead transcription factor is 2-foldincreased in AD BEC. Studies have shown that the AFX forkheadtranscription factor directly regulates apoptosis by suppressing theanti-apoptotic BCL-XL protein (19), that is also down regulated in ADBEC (not shown). Increased levels of BAI2, a p53-target gene homologousto brain-specific angiogenesis inhibitor 1 (20) could modulatepathogenic response of AD BEC during angiogenesis.

Increased brain endothelial expression of tissue transglutaminase T2, anenzyme that catalyzes varepsilon-lysine to gamma glutaminyl isodipeptidebonds, has been reported in AD brains (21). Transglutaminase is involvedin cross-linking of tau and neurofilaments in AD. Our studies in braintissue sections confirmed its endothelial localization (data not shown)suggesting that endothelium may be an important source of increasedtransglutaminase activity in AD brains.

The key translation initiation factor 2 (elF2) gamma subunit (22), and aribosomal protein 37a, encoding a ribosomal component of the 60S subunitwere down regulated in AD BEC, which predicts reduced protein synthesisoverall. AD BEC down regulate plectin, a member of the cytolinkerfamily, which is a stabilizing element of cells against mechanicalstress and is a substrate of caspase 8, that may be required forreorganization of the microfilament system during apoptosis (23). Downregulation of procollagen I-N proteinase, a metalloproteinase thatcleaves amino-propetides in the processing of type I procollagen intocollagen (24) predicts defective matrix processing during AD-mediatedangiogenesis.

It is intriguing that the present analysis revealed that the geneencoding MTG8-related protein MTG16a was significantly up regulated inAD BEC. This gene was originally identified as one of the loci involvedin t(8;21)(q22;q22) chromosomal translocation in acute myeloid leukemia(25). Its role in AD BEC vascular pathology remains to be determined.One or more master key genes may initiate genomic instability in AD BECpossibly by inducing somatic mutations in the endothelium.

It is noteworthy that about 7.5% of dividing early passage subconfluentBEC derived from individuals with Alzheimer's disease had irregularnuclear boundaries, multi-lobed nuclei, and multiple nucleations, whichare characteristic of mitotic catastrophe (Jonathan et al., Curr. Opin.Chem. Biol. 3:77-83, 1999). Cells undergo mitotic catastrophe if theirchromosomal DNA is damaged (e.g., by irradiation). Such cells cannotsuccessfully complete the cell cycle as they cannot enter mitosis, andtherefore they die by mitotic catastrophe. This type of cell death isdifferent from classically-described apoptosis and is characterized bythe inability of the cells to divide (although their nuclei can divide,so typically one finds multinucleation), and they eventually die. Anincreased index of mitotic catastrophe in Alzheimer's disease cells isfound when they are stimulated to grow by growth factors.

Mitotic catastrophe is frequently associated with increased expressionof proteins involved in the initiation and execution of mitosis.Transcriptional profiles suggest significant down regulation of severaltumor suppressor genes including gax (−2-fold; p=0.003), interferoninducible protein 9-27 (−2.2-fold; p=0.04), growth arrest specific gene1 or gas1 (−2.6-fold; p -0.14), AIM-1 (−3.0-fold; p=0.12), c-maf(−3.1-fold; p=0.005), a transcription factor that has the potential toact as tumor suppressor, and increased expression of PISSLRE gene orCDK10, a novel member of cdk family implicated in the G2/M transition(+2.5-fold; p=0.01). Increased activity of MTG-8 may lead to ahyperproliferative response as well.

Thus, mitotic catastrophe could be one possible way to remove damagedbrain endothelial cell from the vascular system in Alzheimer's diseasepatients. On the other hand, brain endothelial cells in Alzheimer'sdisease may activate a cellular program for senescence. Decreasedexpression of gax (1,2) and significantly decreased expression of theantiproliferative interferon-inducible protein 9-27 gene (26)demonstrate that there are cell cycle abnormalities in AD BEC.

Cell death (through mitotic catastrophe followed by apoptosis) andsenescence are two independent responses that could be co-induced bydifferent types of cellular damage (Jonathan et al., Curr. Opin. Chem.Biol. 3:77-83, 1999). In contrast to replicative senescence,stress-induced premature senescence (SIPS) in AD BEC could reflect aprogrammed protective response to cellular stress as in otherage-related diseases (e.g., atherosclerosis, diabetes, and the moregeneral problem of organismic aging).

Significantly slower protein synthesis is suggested by down regulationof some key components, such as elF2 gamma subunit and ribosomal protein37, along with overexpression of transglutaminase TG2, which is likelyto reflect the presence of senescent cells in AD BEC population. Upregulated in BEC of individuals with Alzheimer's disease is cytochromeP450C11 beta, CYP11B1 (+2.5-fold, p=0.005) also increased in senescentHUVEC. It is noteworthy that the AD BEC in the first analysis were fromearly passages that contain approximately 20%-40% of senescent cells. Asshown in Table 12, the molecular and cellular phenotypes of the BECpopulation which contain greater than 85% replicative senescent cellsare very different from mixed AD BEC populations with lower numbers ofsenescent cells, but is similar to the profile of SIPS in control BEC,as discussed below.

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In addition, the expression of other genes was significantly altered inBEC of individuals with Alzheimer's disease as compared to normal agedcontrol (Table 9) (p<0.05): W27720 several genes (+2.3-fold), AL050374(+2.3-fold), J04164 (−2.3-fold), AC005053 (+2.2-fold), A1557295(−2.1-fold), and Y11284 (+2.0-fold); CD2 binding protein recognizes themembrane proximal proline rich tandem repeat on the T-cell receptor CD2,which is involved in cytokine production (AF104222; −2.5-fold, p+0.04);fatty acid desaturase (AL050118; −2.2-fold, p=0.046); ecto-ATPdehydrolase I, endothelial CD39/ectoADPase which has a major 30 role invascular hemostasis by rapidly metabolizing ADP released from platelets,thus preventing further platelet activation and recruitment (AI743406:−2.0-fold, p=0.003); and beta-defensin 2, a cationic peptide crucialcomponent of innate immunity (AF071216; −2.0-fold, p=0.02).

Although changes in some genes did not reach statistical significance inthe first U95A analysis using stringent criteria as described, theystill may be important for better understanding of defectiveangiogenesis. For example, semaphorin IlIl which has a role in extensionand stabilization of vascular networks was down regulated in MBEC ofindividuals with Alzheimer's disease by 2-fold in greater than 85% ofcases. The gene encoding the Dtk tyrosine kinase receptor, abundantlyexpressed in differentiating hematopoietic cells, was up regulated by1.7-fold in greater than 89% of MBEC of individuals with Alzheimer'sdisease confirming loss of differentiated endothelial phenotype inAlzheimer's disease.

In greater than 92% of Alzheimer's disease cases, the gene encoding thelow density lipoprotein receptor related protein-1 (LRP-1) was downregulated by 1.5-fold (p=0.08). LRP-1 activity is down regulated inhuman senescent endothelial cells in athersclerosis (Vasile et al.,FASEB J. 15:458-466, 2001). The LRP-1 protein is down regulated in branvessels in mice during normal aging and in Alzheimer's disease brains,as we previously reported (Shibata et al., J. Clin. Invest.106:1489-1499, 2000). Thus, elevated brain Aβ peptide levels could berelated to down regulation of LRP-1 in brain endothelial cell (Shibataet al., J. Clin. Invest. 106:1489-1499, 2000) that serves as a clearancereceptor for Aβ peptide at the BBB. LRP-1 down regulation could be justa part of a senescent phenotype of Alzheimer's disease brain endothelialcell, but may have a detrimental effect on Aβ accumulation.

Importantly, Aβ40/42 peptides did not produce apoptosis in brainendothelial cell of control or Alzheimer's disease in several assaysthat we have used, confirming that brain endothelium has no enhancedsensitivity to wild type Aβ peptide as described for other cell types.Thus, endothelial changes that have been observed in these studies maybe independent of the presence of Aβ peptide, but the CNS accumulationof Aβ in Alzheimer's disease could be related to an abnormal endothelialphenotype. In support of this hypothesis, we generated new data showingthat incubation of normal or AD BEC with Aβ40/Aβ42 at the levels of 25nM for each peptide does not affect the cell cycle or BEC, and does notaffect their angiogenic potential. Increasing concentrations ofAβ40/Aβ42 up to 500 nM were also without effect and did not induceeither the senescent phenotype in BEC or altered brain capillarymorphogenesis. We also studied BEC isolated from a mouse model of AD,Tg2576sw+/−, with a Swedish APP mutation. BEC isolated from 20 monthsold mice were not senescent and performed normally in an angiogenesis2-D gel assay. Thus, our present findings suggest that cerebrovascularamyloidosis could be secondary to primary cell biology and molecularalterations in BEC, rather than affecting BEC biology. These data areconsistent with previous reports showing that Aβ does not affectendothelial cells (Miravelle et al., J. Biol. Chem. 275:27110-27116,2000), in contrast to its detrimental effects on neurons.

In greater than 65% and greater than 69% of all Alzheimer's diseasecases, respectively, the multiple drug resistance protein-1 and theABCA1 protein, the deficit of which is responsible for Tangier diseaseand familial hypoalphalipoproteinemias, were down regulated by 1.7-foldand 2.6-fold, respectively, thus possibly contributing to brainaccumulation of xenobiotics, bile salts, and/or cholesterol in thoseAlzheimer's disease patients. Down regulation of this gene may reflectcell transformation from a mature differentiated phenotype into afunctionally inferior senescent phenotype.

Significant decreases in production and secretion of neurotrophicfactors in MBEC derived from Alzheimer's disease patients was shown fornerve growth factor (NGF) by ELISA in cell culture supernatants. Reducedexpression of NGF mRNA was confirmed in greater than 80% of Alzheimer'sdisease cases. This correlated with diminished neuritic outgrowth inhippocampal neurons co-cultured with brain endothelial cell derived fromAlzheimer's disease patients. These data are reminiscent of neuriticloss in Alzheimer's disease and an animal model in which intracellularNGF disruption has been achieved (Capsoni et al., Proc. Natl. Acad. Sci.USA 97:6826-6831, 2000).

Additional gene targets were identified in somewhat older AD patientswith more severe pathology than were used in our second analysis usingAffymetrix U133A and U133B chips (Tables 10-11). BEC from these patientsexhibit similar changes in angiogenesis and senescence as seen in FIGS.1-3, 7 and 9-10. Transcription factor E2F that is involved in apoptosiswas increased in AD BEC, as well as cell differentiation factorsneuroglin, E74-like factor 4 and neurogranin, while myelin transcriptionfactor 1 was decreased. As shown in the first analysis, the expressionof PDE-1 was reduced as well as the expression of another member of theankyrin. family, ankyrin 1, and some additional cytoskeletal proteinssuch as myosin VI. in addition to changes in MMP-1, the present analysisshowed that MMP-2 is also altered in AD. Both forms of tarnsglutaminasewere increased as in the first analysis, while another ribosomal proteinpoly(rC)-binding protein 3 was decreased. Several genes involved inlipid metabolism, general metabolism and inflammation were also altered,and several forms of potassium channels and transporter for ferritin. Asignificant number of genes with currently unknown function was alteredon both U133A and U133B arrays and listed in Tables 10-11.

The third analysis shows that 181 genes were altered in the samedirection in RS-AD BEC and young SIPS BEC, suggesting first, that an ADBEC population with a fully-developed replicative senescent (RS)phenotype (greater than 85%) is very different from an early passage ADBEC population containing only a smaller population of senescent cells(20-40%) and second, that our H₂O₂ model of SIPS in young BEC is verysimilar to RS-AD BEC, not just in phenotype (i.e., cellular changes) butalso in terms of affected genes (Table 12). For example, in both models,a total of 43 cell cycle genes and 19 DNA synthesis. genes were downregulated. In addition, several lysosomal/endosomal genes were upregulated or down regulated suggesting a significant disorder oflysosomes in RS AD BEC and SIPS YC BEC. Staining of AD brains in situconfirmed the expected accumulation of products associated with alysosomal storage vascular disorder. Expression of adhesion/matrixproteins was increased including several collagens (e.g., collagen IV),which fits well with the increased thickness of the basement membrane inAD (Miyakawa et al., Virchows Arch. 40:121-129, 1982; Yamada,Neuropathology 20:8-22, 2000). Other findings suggest significantcomparable abnormalities in cell signaling, and expression of severaltranscription factors, and genes involved in inflammation, and a numberof miscellaneous genes all listed in Table 12.

Each of the altered genes alone and/or in combination with other alteredgenes in BEC of individuals with Alzheimer's disease found using theAffymetrix U95A, U133A, and U133B arrays, and/or in RS-AD BEC and SIPSYC BEC, could represent a vascular therapeutic target. Correction of agiven gene's expression may revert and/or contribute to stabilizing anormal brain endothelial phenotype, which ultimately should result incorrection of the cell phenotype.

Altered cellular processes have been identified which can causedefective angiogenesis and an inappropriate senescence. They can beregarded as distinct vascular therapeutic targets in Alzheimer'sdisease: (1) defective differentiation of brain endothelial cells intovascular tubules when stimulated by growth factors and/or otherangiogenic factors; (2) programmed cell death during all stages ofangiogenesis and aberrant response to growth factors resulting inactivation of programmed cell death via a p53-mediated,p38MAPK-mediated, and/or anioxismediated mechanisms early during celldifferentiation; (3) regression in the number of newly formedcapillaries and/or vessels due to genomic instability; (4) silenthyperproliferative slow-growing “cancer-like” disease of brainendothelium similar to acute myeloid leukemia caused by thet(8;21)(q22;q22) translocation; (5) disrupted signaling from the plasmamembrane to the nucleus; (6) dysregulation of one or more genes whichencode transcription factors involved in angiogenesis anddifferentiation; (7) down regulation of one or more tumor suppressors;(8) abnormal gax-integrin αvβ3 and/or αvβ5 signaling; and (9) overexpression of unligated integrins possibly leading to improper signalingthrough integrin-linked kinase.

Senescence of brain endothelium is of pathogenic and clinical relevanceto Alzheimer's vascular disorder and dementia. The present findings alsosuggest that specific vascular-based prophylactic or therapeuticstrategies targeted at presenescent brain endothelial cell can bedeveloped. They can be applied to animal models of Alzheimer's disease,and treatment of patients with Alzheimer's disease or at risk thereof.

The discovery that senescence of cells of the vascular system isimplicated in the development of Alzheimer's dementia presentsopportunities to implement several strategies using our models: e.g., toprovide prophylaxis or therapy in these models including FDA-approveddrugs to inhibit growth dysregulation, prevent senescence, assistsuccessful escape from senescence, prevent mitotic catastrophe and/orapoptosis during escape from senescence, etc.

Changes in cellular phenotype and unstable genotype of brain endotheliummay result in and/or be associated with senescence and/or mitoticcatastrophe which in turn may be caused by a disease-specificAlzheimer's defect in the vascular system. This defect may either reducethe longevity of the vascular system in the brain and/or predispose tostress-induced senescence. The presence of senescent cells in thevascular system can significantly reduce normal physiological functionsof the blood-brain barrier related to molecular exchanges between bloodand brain, and may impair the cerebral blood flow and alter localintravascular hemostasis. Described herein are (1) replicativesenescence of brain endothelial cells in Alzheimer's disease; (2) celldeath of brain endothelium through mitotic catastrophe due to inabilityof cells to enter mitosis; (3) arrest of cells in the G1 phase of thecell cycle and inability of cells to enter the S-phase of the cellcycle; (4) karyokinesis without cytokinesis, or abnormal divisions ofcell nuclei without proper cell division leading to accumulation ofmultinucleated cells; (5) dysregulation of genes encoding keycytoskeletal proteins, translation factors, matrix proteins andtransduction signaling; (6) an in vitro model of Alzheimer's likestress-induced premature senescence (SIPS) with sublethal dose of H₂O₂;(7) molecular events leading to SIPS in human brain endotheliumincluding transient elevation of p53 tumor suppressor, lack of Rbphosphorylation, and inhibition of cyclin-dependent kinase inhibitorsp21^(cIP1) and p16^(INK4a); (8) abnormal capillary morphogenesis fromsenescent cells; (9) potential therapeutic approaches to prevent and/ortreat brain endothelial SIPS and mitotic catastrophe.

In addition, a SIPS model of RS AD BEC suitable for testing differentdrugs has been developed. Studies also suggest significant disorder oflysosomes in senescent cells.

Gene transfer with the candidate genes identified as causingdysregulation of vascular function (e.g., genes listed in Table 9),homeobox gax or mox2, tumor suppressor genes (e.g., gasl,interferon-inducible protein 9-27), transcription factors (e.g., c-MAF,TINUR NGF-B/nurr77), capillary morphogenesis genes (e.g., induction ofSema-3, suppression of BAI1), etc., may be used as described in thisinvention. Expression constructs can be designed to either produce anincrease or decrease in a particular gene product and its cognatepathway as for example, decrease in the case of the gene encodingMTG8-related protein MTG16a. For example, this gene could be regarded asa candidate gene responsible for initiating Alzheimer's disease vasculardisorder. Using either constitutive or drug-controlled vectors,expression of key regulatory genes can be used to reverse or attenuatethe pathogenic process in the microvessel endothelium. Tissue-specificpromoters can be configured into vectors to convey expression to thecell of interest. Repeated application of therapeutic genes is likely tobe needed. Following in vitro studies, gene transfer will be performedex vivo to microvessels and finally in vivo to vessels using differentanimal models. Such techniques have been successfully applied toendothelial cells.

Antisense, ribozyme, RNA interference, and triple helix strategies mayalso be used to inhibit the activity of genes which are up regulated(e.g., the gene encoding MTG8-related protein MTG16a).

Similar approaches could be applied for other targets identified withAffymetrix U133A and U133B arrays (Tables 10-11) in other studiedcohorts of AD patients and controls (Tables 4-7), or for replicativesenescence of BEC AD type and/or SIPS.

High-throughput cell-based assays using fluorescent reporter genereadouts may be developed in several areas for drug screening.Transcription factors that we have discovered to be abnormally regulatedcan be used in reporter gene constructs (e.g., TINUR NGF-B/nur 77 betatype similar to NOT, c-maf). These factors may either have known ciselements through which they transactivate gene expression or SMBselection can be used to deduce the cis elements when they arepreviously unknown. Concatenated cis elements may be placed upstream ofa fluorescent reporter and this construct stably transfected intomammalian cell lines of several types including those of endothelial ornonendothelial origin, and derived from human or animal species.First-order screening of compounds identifies those compounds thateither increase or decrease fluorescence. Second-order screening derivedose-dependent activities for each compound. Third-order screening inour well-characterized cellular models (e.g., MBEC from individuals withAlzheimer's disease) will be followed by in vivo testing in the animalmodels.

FDA approved anti-neoplastic drugs, including alkylating agents (e.g.,cytoxan), nucleoside analogs (e.g., FUdR), or anti-metabolites (e.g.,methotrexate) among others, may be used to control abortive cell growthor other drugs used to treat acute myeloid leukemia havingt(8;21)(q22;q22) (e.g., doxyrubicin applied at a low dose). These arenovel applications for these drugs. Similarly, radiosensitizing agentsfor vascular delivery and retention in the endothelium may be followedby low level external beam X-irradiation, which is used to control anyproliferative disorder leading to dementia (e.g., silenthyperproliferative disorder of brain endothelium). Small therapeuticanti-apoptotic compounds, some of which are FDA approved, may exertanti-apoptotic actions during defective angiogenesis: e.g., SB203580 andSB202190, inhibitors of MAPK p38, molecules that act downstream in thesignaling pathway such as NF_(κ)-B inhibitors that are activated by MAPK(e.g., terolidin-thio-pyridine carbomaleate), molecules that destabilizep53 or enhance its degradation, inhibitors of cysteine-dependentaspartate cleaving proteases (e.g., ZVAD-fmk or peptideAsp-Glu-Val-Asp-al) may restore at 10 least some cell functions byinhibiting multiple regulator proteases, which activate the caspasesignaling pathway, or a particular effector protease (e.g., caspase 3),etc.

Small molecules that correct for impaired intracellular signaling mayalso be used, such as those to block MAPK and signals that are inducedby phosphorylated MAPK, to increase signaling within the GTP/cGMPpathway, to inhibit increased L-3-phophosertine phosphatase activity, orto increase PDE1 and PDE1B1 activity. Candidate compounds includePD98059, an inhibitor of MAPK. Forskolin may have the potential toreestablish impaired signaling due to down regulation of GTPaseactivating proteins (REM), to block activated tyrosine kinase receptors,etc. Anti-oxidants such as GSH and N-acetyl-cysteine, or S-adenosylmethionine may alter the redo state of the cell and alleviate apoptoticsignals.

All references (e.g., articles, books, patents, and patent applications)cited above are indicative of the level of skill in the art and areincorporated by reference.

All modifications and substitutions that come within the meaning of theclaims and the range of their legal equivalents are to be embracedwithin their scope. A claim using the transition “comprising” allows theinclusion of other elements to be within the scope of the claim; theinvention is also described by such claims using the transitional phrase“consisting essentially of” (i.e., allowing the inclusion of otherelements to be within the scope of the claim if they do not materiallyaffect operation of the invention) and the transition “consisting”(i.e., allowing only the elements listed in the claim other thanimpurities or inconsequential activities which are ordinarily associatedwith the invention) instead of the “comprising” term. No particularrelationship between or among limitations of a claim is meant unlesssuch relationship is explicitly recited in the claim (e.g., thearrangement of components in a product claim or order of steps in amethod claim is not a limitation of the claim unless explicitly statedto be so). Thus, all possible combinations and permutations of theindividual elements disclosed herein are intended to be considered partof the invention.

From the foregoing, it would be apparent to a person of skill in thisart that the invention can be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments should be considered only as illustrative, not restrictive,because the scope of the legal protection provided for the inventionwill be indicated by the appended claims rather than by thisspecification. TABLE 1 Alzheimer's disease patients used for U95Aanalyses (12,600 genes) to determine a subset of Alzheimer'sdisease-specific genes CAUSE OF VASCULAR RAD AGE GENDER PMI DEATH RISKFACTORS ANGIOPATHY BRAAK CERAD CDR 001 66 M 5 h Cardiac ArrestAtherosclerosis + V-VI F 3 003 86 F 3 h Respiratory Atherosclerosis +V-VI F 3 Failure 015 67 F 4 h 10 m Respiratory Atherosclerosis + V-VI M4 Failure 20 70 M 5 h Pneumonia None + V-VI M 4 36 80 M 2 h 20 m CardiacArrest Hypertension + V-VI M 4 45 66 F 4 h Pneumonia None + V-VI F 4

TABLE 2 Age-matched controls used for U95A analyses (12,600 genes) todetermine a subset of Alzheimer's disease-specific genes CAUSE OFVASCULAR RAD AGE GENDER PMI DEATH RISK FACTORS ANGIOPATHY BRAAK CERADCDR 13 92 F 7 h Cardiac Arrest Atherosclerosis + 0-I M 0.5 14 88 M 1 h24 m Respiratory Atherosclerosis + 0-I S 0 Arrest 16 64 M 4 h 30 mCardiac Arrest Hypertension, − 0-I 0 0 Atherosclerosis 17 59 F 4 h 30 mStroke None − 0 0 0 38 58 F 5 h 30 m Pulmonary None − 0 0 0 Embolism 3972 M 4 h 20 m Cardiac Arrest Atherosclerosis, − 0-I S 0 MyocardialInfarct

TABLE 3 Young controls used for U95A analysis (12,600 genes) todetermine a subset of Alzheimer's disease-specific genes CAUSE OFVASCULAR RISK RAD AGE GENDER PMI DEATH FACTORS 8 37 F 6 hrs Trauma None12 21 M 6 hrs Trauma None 18 16 F 3 h 30 m Trauma None 19 17 M 3 h 30 mTrauma None 35 26 M 3 h 20 m Trauma None

TABLE 4 Alzheimer's disease patients used for U133A and B analyses(45,000 genes) to determine a subset of Alzheimer's disease-specificgenes CAUSE OF VASCULAR RAD AGE GENDER PMI DEATH RISK FACTORS ANGIOPATHYBRAAK CERAD CDR 001 66 M 5 h Cardiac Arrest Atherosclerosis + V-VI F 3002 88 M 4 h Sepsis Atherosclerosis − V-VI F 3 003 86 F 3 h RespiratoryFailure Atherosclerosis + V-VI F 3 015 67 F 4 h 10 m Respiratory FailureAtherosclerosis + V-VI M 4 20 70 M 5 h Pneumonia None + V-VI M 4 36 91 M2 h 20 m Cardiac Arrest Hypertension + V-VI M 4 037 80 F 3 h 10 mCardiac Arrest Atherosclerosis + V-VI F 5 45 66 F 4 h Pneumonia None +V-VI F 4 122 99 F 3 h 30 m Cardiac Arrest Atherosclerosis + V-VI F 5 12395 F 2 h 30 m GI Bleeding None − V-VI F 3 124 78 M 3 h 30 m BowelObstruction Hypertension + V-VI F 3

TABLE 5 Age-matched controls used for U133A and B analyses (45,000genes) to determine a subset of Alzheimer's disease-specific genes CAUSEOF VASCULAR RAD AGE GENDER PMI DEATH RISK FACTORS ANGIOPATHY BRAAK CERADCDR 13 92 F 7 h Cardiac Arrest Atherosclerosis + 0-I  M 0.5 14 88 M 1 h24 m Respiratory Arrest Atherosclerosis + 0-I  S 0 39 72 M 4 h 20 mCardiac Arrest Atherosclerosis, − 0-I  S 0 Myocardial Infarct 99 74 F 2h 30 m Lung Carcinoma Atherosclerosis + III-IV  M 0 131 84 F 2 h 45 m GIBleeding None −  I-II M 0

TABLE 6 Middle age controls used for U133A and B analyses (45,000 genes)to determine a subset of Alzheimer's disease-specific genes CAUSE OFVASCULAR RAD AGE GENDER PMI DEATH RISK FACTORS ANGIOPATHY BRAAK CERADCDR 16 64 M 4 h 30 m Cardiac Arrest Hypertension, − 0-I 0 0Atherosclerosis 17 59 F 4 h 30 m Stroke None − 0 0 0 38 58 F 5 h 30 mPulmonary None − 0 0 0 Embolism 67 59 M 4 h 30 m Pneumonia None − 0 S 0127 57 F 6 h 40 m Cardiac Arrest Myocardial − 0 S 0 Infarct

TABLE 7 Young controls used for U133A and B analyses (45,000 genes) todetermine a subset of Alzheimer's disease specific genes CAUSE OFVASCULAR RISK RAD AGE GENDER PMI DEATH FACTORS 8 37 F 6 hrs Trauma None18 16 F 3 h 30 m Trauma None 19 17 M 3 h 30 m Trauma None 35 26 M 3 h 20m Trauma None 77 42 F 6 hrs Trauma None

TABLE 8 Patients for study of replicative senescence and stress-inducedsenescence Case PMI Angi- Category # Age Gender (hr) opathy Brakk CERADYC 035 26 M — − ˜ None YC 062 18 F — − ˜ None YC 063 14 F — − ˜ None AMC013 92 F 7 − ˜ None AMC 014 88 M 1 − ˜ None AMC 016 64 M 4.5 − 0-I NoneAMC 017 59 F 4.5 − 0 None AMC 038 58 F 5.5 − 0 None AD 001 66 M 5 + ˜Frequent AD 002 88 M 4 − ˜ Frequent AD 003 86 F 3 +  V-VI Frequent AD015 67 F 4.2 +  V-VI Moderate AD 020 83 M 5 +  V-VI Moderate AD 036 91 M2.4 +  V-VI Moderate AD 037 80 F 3.2 +  V-VI Frequent AD 045 66 F 4 − V-VI Frequent

TABLE 9 A subset of Alzheimer's disease-specific genes in AD BEC vs.age-matched control (AMC) BEC on Affymetrix U95A chips (12,600 genes).The analysis was based on six AD patients, six age-matched controls, andfive young controls. Details about patients are given in Tables 1-3.Changes in gene expression between AD vs. AMC BEC were corrected forchanges in young BEC vs. AMC BEC. GenBank Accession# Gene Name Δ Fold PS77154 TINUR = NGFI-B/nur77 beta-type −7.0 0.047 transcription factorM55153 tissue transglutaminase aka TG2 or tTG 5.8 0.00 AJ001612L-3-phosphoserine-phosphatase 5.3 0.04 homologue L06499 ribosomalprotein L37a −3.8 0.004 AF055376 c-maf −3.1 0.005 U40370 calcium,calmodulin-regulated −3.0 0.015 3′,5′-cyclic nucleotide phophodiesteraseZ54367 plectin −3.0 0.03 AJ003125 procollagen I-N proteinase −2.8 0.049AI381790 novel adipose specific collagen-like factor −2.8 0.04 X55764cytochrome P-45011 beta 2.5 0.005 AF084465 REM −2.6 0.03 L26336 humanheat shock protein 2 −2.5 0.03 AI011896 Nef-associated factor 1 beta 2.50.007 AF104222 CD2BP2 CD2 binding protein −2.5 0.04 L19161 elF-2 gamma−2.5 0.007 X78342 PISSLRE 2.5 0.01 ??????? FK506-binding protein 2.40.008 X52896 dermal fibroblast elastin −2.4 0.04 AB005298 BAI2homologous to brain-specific 2.3 0.03 angiogenesis inhibitor 1 J04164interferon-inducible protein 9-27 −2.2 0.041 Z80776 histone H2A 2.20.041 U13616 ankyrin G (ANK-3) −2.2 0.03 AJ011311 AP endonuclease XTH22.2 0.002 U86078 PDE1B1 calmodulin-stimulated −2.2 0.02phosphodiesterase AL050118 fatty acid desaturase 2 (FADS2) −2.2 0.046AB010419 MTG8-related protein MTG16a 2.1 0.01 NP039256 glial growthfactor 2 2.1 0.01 Q02297 AJ133133 ecto-ATP diphosphohydrolase I 2.1 0.01AI743406 GAX −2.0 0.003 AF071216 beta defensin 2 (HBD2) −2.0 0.02 W27720UNKNOWN 2.3 0.05 AL050374 UNKNOWN 2.3 0.05 J04164 UNKNOWN 2.3 0.05AC005053 whole chromosome 2.2 0.05 AI557295 unknown (BLASTed)-pancreaticcancer p8 −2.1 0.05 Y11284 Parkinson's related; distantly related to 2.00.05 Forkhead transcription factor

TABLE 10 Subset of Alzheimer's disease-specific genes in AD BEC vs.age-matched control (AMC) BEC on Affymetrix U133A chips (22,300 genes).The analysis was based on 11 AD patients, five age-matched controls,five middle-age controls, and five young controls. Details aboutpatients and groups are given in Tables 4-7. Changes in gene expressionbetween AD vs. AMC BEC were corrected for changes in young BEC vs. AMCBEC. Statistical analysis was performed using Bayesian t- test (2-foldratio, signal at 500 expression, and 0.05 Bayesian p-log). Δ Fold P GeneName GenBank Accession# Adhesion −2.06 0.043 osteoblast-specific factor2 D13665 (fasciclin 1-like) Apoptosis −2.02 0.013 myxovirus (influenza)resistance 1, NM_002462 interferon-inducible protein p78 (mouse) CellCycle 2.22 0.023 activator of S phase kinase NM_006716 2.17 0.023 originrecognition complex, subunit 1-like NM_004153 (yeast) 2.25 0.008 MCM4minichromosome maintenance AA604621 deficient 4 (S. cerevisiae) 2.400.008 H2B histone family, member J NM_003524 Differentiation 3.400.00005 neuregulin 1 NM_013959 Immune Response −2.46 0.016immunoglobulin heavy constant mu X95660 −2.21 0.010 ribonuclease L(2′,5′-oligoisoadenylate NM_021133 synthetase-dependent) Ion Channels,Transporters 2.12 0.043 potassium intermediate/small NM_002250conductance calcium-activated channel, subfamily N, member 4 −2.02 0.036phospholamban AW969803 Lipid Metabolism 2.18 0.009 prostaglandin Ereceptor 2 (subtype EP2) NM_000956 53 kDa 3.52 0.040 apolipoprotein BmRNA editing enzyme, NM_004900 catalytic polypeptide-like 3B −2.28 0.015prostaglanding I2 (prostacyclin) synthase NM_000961 Metabolism 6.010.002 transglutaminase 2 (C polypeptide, protein- BC003551glutamine-gamma-glutamyltransferase) −2.40 0.019 phenol sulfotransferaseU37025 2.21 0.024 glycerol kinase X68285 2.33 0.021 similar toacetyl-coenzyme A synthetase; AL049709 similar togamma-glutamyltranspeptidase 2.11 0.008 glutamate decarboxylase 2(pancreatic NM_000818 islets and brain, 65 kDa) Matrix 4.03 0.037 matrixmetalloproteinase 1 (interstitial M_002421 collagenase) SignalTransduction 2.49 0.001 neuroepithelial cell transforming gene 1NM_005863 2.06 0.043 p21 (CDKN1A)-activated kinase 4 NM_005884 3.150.010 neurogranin (protein kinase C substrate, NM_006176 RC3) −2.080.008 AMP-activated protein kinase family NM_014840 member 5 −2.18 0.026FYN binding protein (FYB-120/130) AI633888 −2.04 0.038 phosphodiesterase1A, calmodulin- NM_005019 dependent 2.04 0.034 ADP-ribosylation factordomain protein 1, AF230398 64 kDa 2.22 0.014 G protein-coupled receptor39 AL567376 Structural, Cytoskeleton −2.41 0.040 Arg/Abl-interactingprotein ArgBP2 NM_021069 −3.28 0.027 ankyrin 1, erythrocytic NM_0000372.43 0.010 myosin VI U90236 Transcription Factors, Regulators 2.23 0.001E74-like factor 4 (etx domain transcription NM_001421 factor) 2.65 0.026E2F transcription factor 1 NM_005225 −2.26 0.011 myelin transcriptionfactor 1 M96980 −3.45 0.000 hematopoietic PBX-interacting proteinBP344265 Miscellaneous −2.82 0.007 poly(rC) binding protein 3 NM_020528(RNA-binding) 2.45 0.035 syntaxin binding protein 2 AB002559 −2.16 0.004ferritin H J04755 −3.29 0.002 peptide transporter 3 NM_016582 Unknown−2.10 0.020 chromosome 22 open reading BC001292 frame 4 −2.52 0.005IMAGE: 3460742 AW170549 −2.12 0.041 hypothetical protein FLJ20699NM_017931 −2.20 0.008 secreted protein of unknown AF173937 function 2.000.003 neuropilin (NRP) and tolloid NM_018092 (TLL)-like 2 −2.33 0.005LRP16 protein NM_014067 −2.27 0.034 similar to olfactory receptor,AA731709 family 2; subfamily A, member 4, clone IMAGE: 4424116 −2.150.018 G antigen 3 NM_001473 4.05 0.021 hypothetical protein MGC14258AV717623 −2.32 0.011 FLJ11412 fis, clone AK021474 HEMBA1000876 −2.790.002 moderately similar to ALU7 AI820796 −2.01 0.041 weakly similar toPOL2_MOUSE NM_0185751 retrovirus-related POL polyprotein

TABLE 11 A subset of Alzheimer's disease-specific genes in AD BEC vs.age- matched control (AMC) BEC on Affymetrix U133B chips (22,300 genes).The analysis was based on 11 AD patients, five age-matched controls,five middle-age controls, and five young controls. Details aboutpatients and groups are given in Tables 4-7. Changes in gene expressionbetween AD vs. AMC BEC were corrected for changes in young BEC vs. AMCBEC. Statistical analysis was performed using Bayesian t-test (2-foldratio, signal at 500 expression, and 0.05 Bayesian p-log). GenBank ΔFold P Gene Name Accession# Development −2.07 0.016 limbin AK234305 2.180.010 sema domain, transmembrane AK022831 domain (TM) and cytoplasmicdomain, (semaphorin) 6D Ion Channel, Transporters −2.39 0.022 potassiumchannel, subfamily K, AF110523 member 7 2.11 0.023 adaptor-relatedprotein complex 1, AA480858 sigma 2 subunit 2.02 0.040 potassiuminwardly-rectifying channel, BF111326 subfamily J, member 2 −2.12 0.019highly similar to S72269 ryanodine AA770235 receptor isoform 2Metabolism 2.40 0.024 similar to RIKEN cDNA 2610036L13 BE614410 2.280.038 splicing factor 3a, subunit 1, 120 kDa AI655996 −2.60 0.009 weaklysimilar to activation-induced AI453548 cytidine deaminase 2.63 0.008 E3ubiquitin ligase SMURF2 BF111169 2.01 0.015 peroxisomal acyl-CoAthioesterase 2B AA046424 Proliferation 2.69 0.001 splicing factor,arginine/serine-rich 3 BE927772 Signal Transduction 2.38 0.021 srcfamily associated phosphoprotein N21390 2.21 0.030 2 serine/threoninekinase 11 BE671224 (Peutz-Jeghers syndrome) 2.25 0.015 wingless-typeMMTV integration site AW294903 family, member 7B −3.46 0.000 adenylatecyclase 1 (brain) AK024415 −2.60 0.004 mitogen-activated protein kinaseAK000652 phosphatase x Structural, Cytoskeleton −2.18 0.040Arg/Abl-interacting protein AI659533 2.14 0.010 trichohyalin AI937080Transcription Factors, Regulators 2.15 0.009 MAX gene associatedBF438227 −2.11 0.018 core-binding factor, alpha subunit NM_004349 2,translocated 2.38 0.035 MCM10 minichromosome maintenance AB042719deficient 10 (S. cerevisiae) Zinc Ion Binding −2.34 0.010 tripartitemotif-containing 47 AW249467 Unknown 2.29 0.010 hypothetical proteinMGC2603 BC000209 2.20 0.016 HSPC043 protein BG391217 −2.53 0.005 PRO1953AF130112 2.51 0.007 hypothetical protein FLJ13456 N21008 −2.32 0.013 —AW205640 −2.29 0.010 Homo sapiens cDNA FLJ11862 AU146128 fis, cloneHEMBA1006900 2.14 0.004 clone RP6-45P1 AL035397 2.04 0.029 FLJ30997 fis,clone HLUNG1000104 BF038869 2.02 0.022 — AI445255 2.35 0.041 — AW157450−2.37 0.010 — AI767250 2.39 0.005 — AW291140 −2.31 0.005 — AW291714−2.20 0.028 FLJ32757 fis, clone TEST12001766 AI073559 2.16 0.010moderately similar to hypothetical BF591637 protein FLJ20378 −2.20 0.005— AI417160 −2.19 0.003 — AW511797 3.91 0.001 — AW194766 −2.87 0.001 —AW665538

TABLE 12 Changes in gene expression in AD replicative senescence (RS)BEC and stress-induced premature senescence (SIPS) young control (YC)BEC on Affymetrix U95A chips. Coincidence analysis revealed a subset of181 genes altered in the same direction in RS-AD and SIPS-YC (criteriamore greater than 2-fold change, 500 threshold). For SIPS-YC, YC BEGwere treated with H₂O₂ (300 μM) as in FIG. 8, followed by incubation infresh medium for three days to develop senescent phenotype. For RS-AD,AD BEC were cultured for several passages until greater than 85% ofcells became senescent (i.e., β-gal positive, enlarged morphology). SIPSYC BEG (n = 4) were compared with untreated YC BEC (n = 5) and RS-AD BECwere compared with early passage AD BEC; n = 3 per group. Acc. # SIPS-YCRS-AD Cell cycle p16INK4 U26727 2.37 2.76 growth-arrest-specific protein(gas) L13720 3.83 3.22 forkhead box M1 U74612 −3.08 −4.42 p55CDC mRNAU05340 −4.76 −5.17 mitotic checkpoint kinase Mad3L AF053306 −3.53 −5.11kinesin-like spindle protein HKSP U37426 −3.84 −4.40 kinesin-relatedprotein D14678 −2.92 −2.97 kinesin family member 14 D26361 −4.23 −2.64mitotic kinesin-like protein-1 X67155 −2.09 −3.53 mitoticcentromere-associated kinesin U63743 −3.85 −3.58 lamin B1 L37747 −3.14−4.57 mitotic checkpoint kinase Bub1 AF053305 −3.59 −4.12serine/threonine kinase (STK-1) AF015254 −2.51 −5.04 serine/threoninekinase (BTAK) AF011468 −3.20 −4.86 CDC28 protein kinase 1 AA926959 −2.63−2.14 activator of S phase Kinase AB028069 −2.62 −2.59 CKS1 proteinhomologue X54942 −2.97 −4.12 CDC2-related protein kinase M68520 −2.06−2.79 CDC2 X05360 −2.71 −4.26 CDC6-related protein U77949 −5.91 −7.27cyclin E2 AF091433 −2.38 −2.89 cyclin F Z36714 −2.04 −2.08 cyclin B2AL080146 −2.30 −5.62 cyclin B M25753 −3.86 −6.32 cyclin A X51688 −3.32−2.89 Ki-67 antigen X65550 −2.63 −4.86 Mad2 U65410 −3.07 −4.08 MAD2protein AJ000186 −2.17 −3.23 ZW10 interactor Zwint AF067656 −3.81 −3.94apoptosis inhibitor surviving U75285 −2.59 −2.50 HOX11L1 gene AJ002607−3.60 −3.41 B-myb X13293 −2.28 −2.95 centromere-associated protein,CENP-E Z15005 −3.42 −4.37 centromere protein-A (CENP-A) FU14518 −3.13−3.87 TTK tyrosine kinase M86699 −3.02 −4.26 BRCA1-associated RINGdomain protein U76638 −2.36 −4.23 polo-like serine/threonine kinaseU01038 −2.67 −4.23 fls353 AB024704 −3.53 −3.55 pituitarytumor-transforming 1 AA203476 −3.98 −4.63 barren homolog D38553 −2.18−2.83 discs, large homolog 7 D13633 −2.11 −5.00retinoblastoma-associated protein HEC AF017790 −3.83 −4.62cyclin-selective ubiquitin carrier protein FU73379 −4.93 −7.08 DNAreplication/repair DNA topoisomerase ∥ J04088 −2.26 −4.23 topoisomerase∥ alpha AI375913 −2.33 −5.82 P1-Cdc46 X74795 −2.49 −3.22 P1cdc47 D55716−4.03 −3.31 thymidylate synthase X02308 −2.66 −3.30 thymidylate synthaseD00596 −3.18 −4.40 replication factor C, 37-kDa subunit mRNA M87339−2.32 −2.97 H2A.X mRNA encoding histone H2A.X X14850 −2.49 −2.80 histone(H2A.Z) M37583 −2.05 −2.38 thymidine kinase M15205 −3.63 −4.84 thymidinekinase K02581 −2.85 −5.04 M1 subunit of ribonucleotide reductase X59543−2.83 −4.59 chromosome-associated polypeptide-C AB019987 −2.24 −3.16polymerase (DNA directed), epsilon AL080203 −3.06 −2.70 HMG-2 X62534−2.88 −3.50 zeste homolog 2 (EZH2) U61145 −2.54 −3.89 ribonuclease H |large subunit Z97029 −3.87 −3.03 Rad2 nuclease −2.21 −2.19 dihydrofolatereductase J00140 −4.08 −4.05 Lysosomal/endosomal lysosome-associatedmembrane protein-2 X77196 2.70 2.40 lysosomal sialoglycoprotein D126762.48 2.57 glucosamine-6-sulphatase Z12173 4.02 2.81 carboxypeptidase DU65090 2.42 2.77 soluble PLA2 receptor U17034 2.49 4.38 plasma glutamatecarboxypeptidase AI796048 2.72 2.03 Cell signaling Ins(1, 3, 4,5)P4-binding protein X89399 2.40 2.74 G alpha subunit L01694 2.69 2.37hR-PTPu gene for protein tyrosine phosphatase X58288 3.04 2.45phospholipase 3D U60644 2.03 3.31 Sh3 domain YSC-like 1 AL050373 2.942.17 GABA(A) receptor-associated protein like 1 W28281 2.20 2.13 Ndrprotein kinase Z35102 2.44 2.04 orphan G protein-coupled receptor HG38AF062006 −2.50 −2.03 ADP-ribosylation factor (hARF6) M57763 −2.98 −2.08oncoprotein 18/stathmin M31303 −2.59 −2.70 maternal embryonic leucinezipper kinase (MELK) D79997 −2.76 −3.38 protein tyrosine phosphatase(CIP2) L25876 −3.34 −6.52 Transcriptional regulation transcriptionelongation factor S-II D50495 2.44 2.14 FUS-CHOP protein fusion S621384.64 3.76 p8 protein W47047 4.30 5.51 zinc-finger protein (bcl-6) U001152.27 2.03 brachyury variant B (TBX1) AF012131 2.01 2.61 HOX3D gene forhomeoprotein X61755 −2.07 −2.25 ribonucleoprotein H1 W28483 −2.35 −2.39CDC-like kinase M59287 2.16 2.06 leucine-rich acidic nuclear proteinlike AA913812 −2.11 −2.14 transcription factor RTEF-1 U63824 −2.12 −2.39transactivator protein (CREB) M27691 −2.01 −2.03 Extracellularmatrix/adhesion molecule reversion-inducing-cysteine-rich protein withkazal AA099265 2.39 3.80 motifs (RECK) microfibril-associatedglycoprotein 4 L38486 5.46 13.17 lysyl oxidase (LOX) L16895 2.10 2.62collagenase type IV M55593 3.44 2.50 alpha-1 type XVI collagen M926422.92 3.18 alpha-5 collagen type IV M58526 2.91 2.21 integrin bindingprotein Del-1 U70312 4.29 3.40 integrin alpha-2 subunit X17033 3.56 2.79AICL (activation-induced C-type lectin) X96719 2.49 2.46cartilage-associated protein (CASP) AJ006470 2.30 2.25 fibulin-5AF093118 2.45 3.59 tetranectin/plasminogen-binding protein X64559 3.003.84 B-cam/Lutheran blood group glycoproteins X80026 −2.31 −2.16fibulin-1 B X53742 −2.54 −2.56 intracellular hyaluronic acid bindingprotein AF032862 −3.96 −4.30 Transporter ATP-binding cassette,sub-family A (ABC1) AB028985 3.16 2.51 ATPase, Class V AI478147 2.682.39 calcium channel alpha-2b subunit M76559 2.70 2.31 Na, K-ATPase beta2 subunit AF007876 −2.26 −2.05 Inflammation TGF-betaIIR alpha D506832.60 2.07 transforming growth factor-beta 1 binding protein M34057 2.412.16 latent transforming growth factor-beta binding protein Z37976 2.003.55 FK506 binding protein 9 AA487755 2.25 2.11 importin alpha 7 subunitAF060543 −2.38 −2.05 nuclear localization sequence receptor hSRP1 alphaU28386 −2.69 −3.84 Cell Inflammation immunoglobulin-like transcript 7AF041261 −3.00 −2.10 Miscellaneous secretogranin ∥ M25756 12.08 3.41heat shock 20-kDa protein AI093511 7.35 2.10 semaphorin E AB000220 4.653.50 propionyl-CoA carboxylase alpha-chain X14608 2.05 3.27hydroxysteroid dehydrogenase D17793 3.18 6.02 autotaxin L35594 2.86 6.13spermidine/spermine N1-acetyltransferase AL050290 2.56 5.80 glutaminaseAB020645 2.33 2.26 thioredoxin interacting protein S73591 2.07 2.99beta-mannosidase U60337 2.28 3.21 ubiquitin-conjugating enzyme UbcH2Z29331 3.47 4.14 Synaptosomal-associated protein 23B Y09568 2.29 2.03sorting nexin 13 AB018256 2.30 2.94 hepatic dihydrodiol dehydrogenaseU05861 3.97 4.79 DEAD-box protein p72/RNA helicase U59321 3.37 3.55Membrane cofactor protein X59408 3.01 2.00 complement factor H X075232.22 2.70 cleavage signal 1 protein M61199 2.59 2.37 Mel-18 protein/zincfinger protein D13969 2.13 2.14 amyotrophic lateral sclerosis 2 AB0111212.01 2.04 epithelial membrane protein-2 U52100 −2.16 −2.47 dermatopontinZ22865 −2.32 −2.44 Alstrom syndrome 1 (ALMS1) R40666 −2.55 −2.07coiled-coil related protein DEEPEST AF063308 −2.71 −4.20 translocase ofouter mitochondrial membrane AF043250 −2.00 −2.17 isopeptidase T-3U75362 −2.14 −2.07 N-acetyltransferase-8 AB013094 −2.09 −2.45Opa-interacting protein OIP5 AF025441 −2.22 −3.45 HPV16 E1 proteinbinding protein U96131 −2.76 −3.41 dopamine D2 receptor X51362 −3.40−2.11 NB thymosin beta D82345 −3.06 −5.32 cytosolic serinehydroxymethyltransferase L11931 −2.05 −3.06 glyceraldehyde-3-phosphatedehydrogenase U34995 −11.38 −2.30

1. A method of assaying for vascular dysfunction in a subject affectedby or at risk for a neurodegenerative disorder or another cognitiveimpairment, said method comprising determining whether there isinappropriate senescence and/or defective angiogenesis in at leastendothelium of the subject or cells derived from endothelium of thesubject.
 2. The method according to claim 1, wherein said subject isaffected by or at risk for Alzheimer's disease.
 3. The method accordingto claim 1, wherein said subject is human.
 4. The method according toclaim 1 which further comprises obtaining endothelial cells from saidsubject and culturing said cells to provide derived cells, whereininappropriate senescence and/or defective angiogenesis in at least saidderived cells is indicative of vascular dysfunction.
 5. The methodaccording to claim 1, wherein there is at least (a) abnormal response byendothelial cells to angiogenic signaling; (b) anoikis, apoptosis, orprogrammed cell death; (c) mitotic catastrophe; (d) a storage disorder,or (e) a combination thereof.
 6. The method according to claim 1,wherein there is at least (a) defective differentiation of endothelialcells, (b) defective fusion of capillaries or vessels, (c) inappropriateregression of capillaries or vessels, or (d) a combination thereof.
 7. Amethod of treating a neurodegenerative disorder or another cognitiveimpairment, said method comprising administration of a drug or othertreatment which at least (a) causes neovascularization, (b) reducesdefective angiogenesis, (c) reduces defective capillary morphogenesis,(d) reduces senescence, (e) reduces mitotic catastrophe, or (f) acombination thereof in brain and/or vascular system of a subject toprovide therapy and/or prevention.
 8. A method of treating aneurodegenerative disorder or another cognitive impairment, said methodcomprising administration of a drug or other treatment which at least(a) reduces vascular dysfunction or (b) normalizes expression of one ormore genes whose regulation is dysregulated in brain and/or vascularsystem of a subject to provide therapy and/or prevention.
 9. The methodaccording to claim 7, wherein said neurodegenerative disorder or anothercognitive impairment is Alzheimer's disease.
 10. The method according toclaim 7, wherein said subject is human.
 11. The method according toclaim 7 further comprising assessing improved cognitive function of saidsubject after treatment.
 12. The method according to claim 7 furthercomprising monitoring treatment by assessing cerebral blood flow orblood-brain barrier function.
 13. A method of determining effectivenessof a drug or other treatment of a neurodegenerative disorder or anothercognitive impairment, said method comprising: (a) applying the drug orother treatment to a subject or an endothelial cell culture; (b)assaying for vascular dysfunction which is not caused by amyloid; and(c) selecting said drug or other treatment if there is reversal ofvascular dysfunction as potentially effective for said neurodegenerativedisorder or another cognitive impairment.
 14. The method according toclaim 13, wherein at least angiogenesis or senescence is assayed asindicative of said vascular dysfunction.
 15. The method according toclaim 13, wherein said subject is a nonhuman animal model.
 16. Themethod according to claim 15, wherein said nonhuman animal model istransgenic for a disease-specific gene.
 17. The method according toclaim 15, wherein said nonhuman animal model has a knock-in or knock-outmutation for a disease-specific gene.
 18. A drug or other treatmentselected by the method according to claim
 13. 19. A method ofdetermining whether cells manifest a phenotype indicative of aneurodegenerative disorder or another cognitive impairment comprisingassessing at least (a) angiogenesis, (b) capillary morphogenesis, (c)differentiation of or signal transduction in endothelial cells, (d) DNAdamage or repair, (e) mitotic catastrophe, (f) senescence, (g) lysosomefunction, or (h) a combination thereof.
 20. The method according toclaim 19, wherein said neurodegenerative disorder or another cognitiveimpairment is Alzheimer's disease.
 21. The method according to claim 19,wherein said cells are derived from a human.
 22. The method according toclaim 19, wherein said cells are derived from a nonhuman animal.
 23. Themethod according to claim 19 which further comprises selecting a drug orother treatment as potentially effective for therapy and/or preventionby said drug's or other treatment's ability to reverse said phenotypeindicative of a neurodegenerative disorder or another cognitiveimpairment.
 24. A drug or other treatment selected by the methodaccording to claim
 23. 25. A method of producing stress-inducedpremature senescent (SIPS) endothelial cells to provide an in vitromodel for a neurodegenerative disorder or another cognitive impairment,said method comprising: (a) providing a culture of endothelial cells,(b) treating said culture with hydrogen peroxide to induce senescence,and (c) isolating SIPS endothelial cells from said treated culture. 26.SIPS endothelial cells produced by the method according to claim 25.